Human cord blood as a source of neural tissue repair of the brain and spinal cord

ABSTRACT

The present invention relates to the use of umbilical cord blood cells from a donor or patient to provide neural cells which may be used in transplantation. The isolated cells according to the present invention may be used to effect autologous and allogeneic transplantation and repair of neural tissue, in particular, tissue of the brain and spinal cord and to treat neurodegenerative diseases of the brain and spinal cord.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of currently pending U.S.Nonprovisional application Ser. No. 13/647,934, filed Oct. 9, 2012,which is a divisional of U.S. Pat. No. 8,309,352 issued Nov. 13, 2012,which is a divisional of U.S. Pat. No. 7,160,724 issued Jan. 9, 2007,which claims priority to expired U.S. Provisional Patent Application60/269,238 filed Feb. 16, 2001 and expired U.S. Provisional PatentApplication 60/188,069 filed Mar. 9, 2000, the contents of each of whichare herein incorporated by reference.

FIELD OF INVENTION

The present invention relates to the use of human umbilical cord bloodand/or mononuclear cell fragment, thereof from a donor or patient toprovide neural cells for use in transplantation. The isolated cellsaccording to the present invention may be used to effect transplantationand repair of neural tissue, in particular, tissue of the brain andspinal cord and to treat neurodegenerative diseases and injury of thebrain and spinal cord.

BACKGROUND OF THE INVENTION

Neurobiologists believe that the neurons in the adult brain and spinalcord are impossible to rebuild once they are damaged. Thus, scienceprovided little hope to patients suffering from brain and spinal cordinjury or from neurodegenerative diseases such as Alzheimer's diseaseand Parkinson's disease, among a number of others. Parkinson's andAlzheimer's diseases are examples of neurodegenerative diseases whichare thought to be untreatable.

Parkinson's disease (PD), is a disorder of middle or late life, withvery gradual progression and a prolonged course. HARRISON'S PRINCIPLESOF INTERNAL MEDICINE, Vol. 2, 23d ed., Ed by Isselbacher, Braunwald,Wilson, Martin, Fauci and Kasper, McGraw-Hill Inc., New York City, 1994,pg. 2275. The most regularly observed changes in patients withParkinson's disease have been in the aggregates of melanin-containingnerve cells in the brainstem (substantia nigra, locus 20 coeruleus),where there are varying degrees of nerve cell loss with reactive gliosis(most pronounced in the substantia nigra) along with distinctiveeosinophilic intracytoplasmic inclusions. (Id. at 2276). In its fullydeveloped form, PD is easily recognized in patients, where stoopedposture, stiffness and slowness of movement, fixity of facialexpression, rhythmic tremor of the limbs, which subsides on activewilled movement or complete relaxation, are common features. Generally,accompanying the other characteristics of the fully developed disorderis the festinating gait, whereby the patient, progresses or walks withquick shuffling steps at an accelerating pace as if to catch up with thebody's center of gravity. (Id. at 2276).

The treatment of Parkinson's disease pharmacologically with levodopacombined with stereotactic surgery has only represented a partial cure,at best. (Id. at 2277). Underlying much of the treatment difficulty isdirected to the fact that none of these therapeutic measures has aneffect on the underlying disease process, which consists of neuronaldegeneration. Ultimately, a point seems to be reached where pharmacologycan no longer compensate for the loss of basal ganglia dopamine. (Id.).

Alzheimer's Disease (AD) is caused by a degenerative process in thepatient which is characterized by progressive loss of cells from thebasal forebrain, cerebral cortex and other brain areas. Acetylcholinetransmitting neurons and their target nerves are particularly affected.Senile plaques and neurofibrillary tangles are present. Pick's diseasehas a similar clinical picture to Alzheimer's disease but a somewhatslower clinical course and circumscribed atrophy, mainly affecting thefrontal and temporal lobes. One animal model for Alzheimer's disease andother dementias displays hereditary tendency toward the formation ofsuch plaques. It is thought that if a drug has an effect in the model,it also may be beneficial in at least some forms of Alzheimer's andPick's diseases. At present, there are palliative treatments but nomeans to restore function in Alzheimer's patients.

A group of related neuronal degenerative disorders is characterized byprogressive ataxia due to degeneration of the cerebellum, brainstem,spinal cord and peripheral nerves, and occasionally the basal ganglia.Many of these syndromes are hereditary; others occur sporadically. Thespinocerebellar degenerations are logically placed in three groups:predominantly spinal ataxias, cerebellar ataxias and multiple-systemdegenerations. To date there are no treatments. Friedrich's ataxia isthe prototypical spinal ataxia whose inheritance is autosomal recessive.The responsible gene has been found on Chromosome 9. Symptoms beginbetween ages of 5 and 15 with unsteady gait, followed by upper extremityataxia and dysarthria. Patients are flexic and lose large-fiber sensorymodalities (vibration and position sense). Two other diseases havesimilar symptoms: Bassen-Kornzweig syndrome (αβ-lipoproteinemia andvitamin E deficiency) and Refsom's disease (phytanic acid storagedisease). Cerebellar cortical degenerations generally occur between ages30 and 50. Clinically only signs of cerebellar dysfunction can bedetected, with pathologic changes have been reported. Similardegeneration may also be associated with chronic alcoholism. Inmultiple-system degenerations, ataxia occurs in young to middle adultlife in varying combinations with spasticity and extrapyramidal,sensory, lower motor neuron and autonomic dysfunction. In some families,there may also be optic atrophy, retinitis pigmentosa, opthalmoplegiaand dementia.

Another form of cerebellar degeneration is paraneoplastic cerebellardegeneration that occurs with certain cancers, such as oat cell lungcancer, breast cancer and ovarian cancer. In some cases, the ataxia mayprecede the discovery of the cancer by weeks to years. Purkinje cellsare permanently lost, resulting in ataxia. Even if the patient ispermanently cured of the cancer, their ability to function may beprofoundly disabled by the loss of Purkinje cells. There is no specifictreatment.

Strokes also result in neuronal degeneration and loss of functionalsynapses. Currently there is no repair, and only palliation andrehabilitation are undertaken.

Neurotransplantation has been used to explore the development of thecentral nervous system and for repair of diseased tissue in conditionssuch as Parkinson's and other neurodegenerative diseases. Theexperimental replacement of neurons by direct grafting of fetal tissueinto the brain has been accomplished in small numbers of patients inseveral research universities (including the University of SouthFlorida); but so far, the experimental grafting of human fetal neuronshas been limited by scarcity of appropriate tissue sources, logisticproblems, legal and ethical constraints, and poor survival of graftedneurons in the human host brain. One method replaces neurons by usingbone marrow stromal cells as stem cells for non-hematopoietic tissues.Marrow stromal cells can be isolated from other cells in marrow by theirtendency to adhere to tissue culture plastic. The cells have many of thecharacteristics of stem cells for tissues that can roughly be defined:as mesenchymal, because they can be differentiated in culture intoosteoblasts, chondrocytes. adipocytes, and even myoblasts. Thispopulation of bone marrow cells (BMSC) have also been used to preparedendritic cells, (K. Inaba, et al., J Experimental Med. 176: 1693-1702(1992)) which, as the name implies, have a morphology which might beconfused for neurons. Dendritic cells comprise a system ofantigen-presenting cells involved in the initiation of T cell responses.The specific growth factor, which stimulates production of dendriticcells, has been reported to be granulocyte/macrophage colony-stimulatingfactor 30 (GM-CSF). K. Inaba, et al., J Experimental Med. 176: 1693-1702(1992).

Work has recently been performed using stem cells obtained from bonemarrow to provide neural cells which can be used in neuronaltransplantation. See WO 99/56759. This patent represented theculmination of more than 130 years of work in the use of bone marrowstem cells for non-hematopoietic uses.

Several groups of investigators since 1990 have attempted to preparemore homogenous populations of stem cells from bone marrow. For example,U.S. Pat. No. 5,087,570, issued Feb. 11, 1992, discloses how to isolatehomogeneous mammalian hematopoietic stem cell compositions. Concentratedhematopoietic stem cell compositions are substantially free ofdifferentiated or dedicated hematopoietic cells. The desired cells areobtained by subtraction of other cells having particular markers. Theresulting composition may be used to provide for individual or groups ofhematopoietic lineages, to reconstitute stem cells of the host, and toidentify an assay for a wide variety of hematopoietic growth factors.

U.S. Pat. No. 5,633,426 issued May 27, 1997, is another example of thedifferentiation and production of hematopoietic cells. Chimericimmunocompromised mice are given human bone marrow of at least 4 weeksfrom the time of implantation. The bone marrow assumed the normalpopulation of bone marrow except for erythrocytes. These mice with humanbone marrow may be used to study the effect of various agents on theproliferation and differentiation of human hematopoietic cells.

U.S. Pat. No. 5,665,557, issued Sep. 9, 1997, relates to methods ofobtaining concentrated hematopoietic stem cells by separating out anenriched fraction of cells expressing the marker CDw 109. Methods ofobtaining compositions enriched in hematopoietic megakaryocyteprogenitor cells are also provided. Compositions enriched for stem cellsand populations of cells obtained therefrom are also provided by theinvention. Methods of use of the cells are also included.

U.S. Pat. No. 5,453,505 issued on Jun. 5, 1995, is yet another method ofdifferentiation. Primordial tissue is introduced into immunodeficienthosts, where the primordial tissue develops and differentiates. Thechimeric host allows for investigation of the processes and developmentof the xenogeneic tissue, testing for the effects of various agents onthe growth and differentiation of the tissue, as well as identificationof agents involved with the growth and differentiation.

U.S. Pat. No. 5,753,505 issued May 19, 1998, provides an isolatedcellular composition comprising greater than about 90% mammalian,non-tumor-derived, neuronal progenitor cells which express aneuron-specific marker and which can give rise to progeny which candifferentiate into neuronal cells. Also provided are methods of treatingneuronal disorders utilizing this cellular composition.

U.S. Pat. No. 5,759,793 issued Aug. 6, 1996, provides a method for boththe positive and negative selection of at least one mammalian cellpopulation from a mixture of cell populations utilizing a magneticallystabilized fluidized bed. One application of this method is theseparation and purification of hematopoietic cells. Target cellpopulations include human stem cells.

U.S. Pat. No. 5,789,148 issued Aug. 4, 1998, discloses a kit,composition and method for cell separation. The kit includes acentrifugable container and an organosilanized silica particle-basedcell separation suspension suitable for density gradient separation,containing a polylactam and sterilized by treatment with ionizingradiation. The composition includes a silanized silica particle-basedsuspension for cell separation which contains at least 0.05% of apolylactam. and preferably treated by ionizing radiation. Also disclosedis a method of isolating rare blood cells from a blood cell mixture.

Within the past several years, mesenchymal stem cells (MSCs) have beenexplored as vehicles for both cell therapy and gene therapy. The cellsare relatively easy to isolate from the small aspirates of bone marrowthat can be obtained under local anesthesia: they are also relativelyeasy to expand in culture and to transfect with exogenous genes.Prockop, D. J. Science 26: 71-74 (1997). Therefore, MSCs appear to haveseveral advantages over hematopoietic stem cells (HMCs) for use in genetherapy. The isolation of adequate numbers of HSCs requires largevolumes of marrow (1 liter or more), and the cells are difficult toexpand in culture. (Prockop, io D. J. (ibid.)).

There are several sources for bone marrow tissue, including thepatient's own bone marrow, that of blood relatives or others with MHCmatches and bone marrow banks. There are several patents that encompassthis source. U.S. Pat. No. 5,476,997 issued May 17, 1994, discloses amethod of producing human bone marrow equivalent. A human hematopoieticsystem is provided in an immunocompromised mammalian host, where thehematopoietic system is functional for extended periods of time. In thismethod, human fetal liver tissue and human fetal thymus are introducedinto a young immunocompromised mouse at a site supplied with a vascularsystem, whereby the fetal tissue results in formation of functionalhuman bone marrow tissue.

Human fetal tissue also represents a source of implantable neurons, butits use is quite controversial. U.S. Pat. No. 5,690,927 issued Nov. 25,1997, also utilizes human fetal tissue. Human fetal neuro-derived celllines are implanted into host tissues. The methods allow for treatmentof a variety of neurological disorders and other diseases.

U.S. Pat. No. 5,753,491, issued May 19, 1998, discloses methods fortreating a host by implanting genetically unrelated cells in the host.More particularly, the present invention provides human fetalneuro-derived cell lines, and methods of treating a host by implantationof these immortalized human fetal neuro-derived cells into the host. Onesource is the mouse, which is included in the U.S. Pat. No. 5,580,777issued Dec. 3, 1996. This patent features a method for the in vitroproduction of lines of immortalized neural precursor cells, includingcell lines having neuronal and/or glial cell characteristics, comprisesthe step of infecting neuroepithelium or neural crest cells with aretroviral vector carrying a member of the myc family of oncogenes.

U.S. Pat. No. 5,753,506 issued May 19, 1998, reveals an in vitroprocedure by which a homogeneous population of multipotential precursorcells from mammalian embryonic neuroepithelium (CNS stem cells) isexpanded up to 10-fold in culture while maintaining their multipotentialcapacity to differentiate into neurons, oligodendrocytes, andastrocytes. Chemical conditions are presented for expanding a largenumber of neurons from the stem cells. In addition, four factors—PDGF,CNTF, LIF, and T3—have been identified which, individually, generatesignificantly higher proportions of neurons, astrocytes, oroligodendrocytes. These procedures are intended to permit a large-scalepreparation of the mammalian CNS stem cells, neurons, astrocytes, andoligodendrocytes. These cells are proposed as an important tool for manycell- and gene-based therapies for neurological disorders. Anothersource of stem cells is that of primate embryonic stem cells. U.S. Pat.No. 5,843,780 issued Dec. 1, 1998, utilizes these stem cells. A purifiedpreparation of stem cells is disclosed. This preparation ischaracterized by the following cell surface markers: SSEA-I (−); SSEA-3(+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In oneembodiment, the cells of the preparation have normal karyotypes andcontinue to proliferate in an undifferentiated state after continuousculture for eleven months. The embryonic stem cells lines are alsodescribed as retaining the ability to form trophoblasts and todifferentiate into tissues derived from all three embryonic germ layers(endoderm, mesoderm and ectoderm). A method for isolating a primateembryonic stem cell line is also disclosed in the patent.

There is substantial evidence in both animal models and human patientsthat neural transplantation is a scientifically feasible and clinicallypromising approach to the treatment of neurodegenerative diseases andstroke as well as for repair of traumatic injuries to brain and spinalcord. Nevertheless, alternative cell sources and novel strategies fordifferentiation are needed to circumvent the numerous ethical andtechnical constraints that now limit the widespread use of neuraltransplantation. In short, there is a need for further development ofreadily available reliable sources of neural cells for transplantation.

The use of umbilical cord blood for use in hematopoietic reconstitutionhas been around since the work of Ende in the early 1970's. Becauseumbilical cord blood is rich in hematopoietic precursors, including stemcells, it represents a good source of cells for hematopoieticreconstitution. To date, however, little work has been done on usingpluripotential stem cells or related neural precursors which are foundin umbilical cord blood for neuronal transplantation perhaps because ofthe failure to realize the viable source of neuronal precursors whichcan be found in umbilical cord blood.

Human cord and placental blood provides a rich source of hematopoieticstem cells. On the basis of this finding, umbilical cord blood stemcells have been used to reconstitute hematopoiesis in children withmalignant and nonmalignant diseases after treatment with myeloablativedoses of chemoradiotherapy. Sirchia and Rebulla, 1999 Haematologica84:738-47. Early results show that a single cord blood sample providesenough hematopoietic stem cells to provide short- and long-termengraftment, and that the incidence and severity of graft-versus-hostdisease has been low even in HLA-mismatched transplants. These results,together with the previous discovery that bone marrow cells contain stemcells capable of differentiating into neurons and glia, led to thepresent invention which uses cord blood or mononuclear cell fractionsthereof to repair neuronal damage in brain and spinal cord.Sanchez-Ramos, et al. 1998. Movement Disorders 13(s2): 122 andSanchez-Ramos, et al., (2000) Exp. Neurol.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a novel source ofpluripotent stem and/or progenitor cells which can be readilydifferentiated into neuronal and glial cells to be used intransplantation in the brain and spinal cord of a patient and for thetreatment of neurodegenerative diseases.

It is an additional object of the invention to provide pharmaceuticalcompositions comprising effective amounts or concentrations of neuralcells for use in transplantation and methods for treatingneurodegenerative diseases, or brain or spinal cord injuries or damage.

It is another object of the invention to provide methods for isolatingand inducing differentiation of pluripotent stem and/or progenitor cellsinto neuronal and glial cells which can be used in transplantationprocedures or for the treatment of neurodegenerative diseases.

It is a further object to provide a method of treating neurodegenerativediseases and spinal cord/brain injury using neural and/or neuronaland/or glial cells derived from umbilical cord blood.

It is yet a further object of the invention to provide a method oftransplanting neural and/or neuronal and/or glial cells derived fromumbilical cord blood in order to repair damaged organs of a patient'snervous system such as the brain and spinal cord.

These and/or other objects of the invention may be readily gleaned fromthe description of the invention which follows.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected discovery that fresh orreconstituted umbilical cord blood or a mononuclear cellular fractionthereof is a novel source of cells which can be differentiated intoneural cells and/or neuronal tissue and used for neuronaltransplantation (autografting as well as allografting), thus obviatingthe need to use pooled neuronal fetal tissue or bone marrow tissue,which is often hard to obtain. Thus, the present invention may be usedfor autografting (cells from an individual are used in that sameindividual), allografting (cells from one person are used in anotherperson) and xenografting (transplantation from one species to another).In this aspect of the present invention, it has unexpectedly beendiscovered that umbilical cord blood from neonates or fetuses comprisescells which may be induced to become neurons in vitro and in vivo. Thesecells may be used in autologous or allogeneic transplantation (grafting)procedures to improve neurological deficit and to effect transplantationand repair of neural/neuronal tissue, in particular, tissue of the brainand spinal cord and to treat neurodegenerative diseases of the brain andspinal cord.

In one aspect, according to the present invention, umbilical cord bloodderived neural cells are suitable for grafting into a patient's brain orspinal cord. These neural cells may be purified and/or incubated with adifferentiation agent by any one or more of the methods otherwisedescribed in the present specification or alternatively, these cells maybe obtained from crude mononuclear cell fractions of umbilical cordblood and used directly without further purification or differentiation.In other aspects, umbilical cord blood may be used without furtherpurification.

In another aspect of the present invention, there is presented a methodfor obtaining neural cells from umbilical cord blood, the methodcomprising the steps of obtaining umbilical cord blood, selectingumbilical cord pluripotential stem cells or progenitor cells which areneural precursor cells and incubating the umbilical cord stem cells orprogenitor cells with a differentiation agent to change the phenotype ofthe cells to produce a population of neural cells which are capable ofbeing transplanted. The steps of the method may also be changed suchthat all of the cells (for example, from an umbilical blood sample or amononuclear cell fraction thereof) are incubated with a differentiationagent prior to separation of the neural phenotype cells.

The method of the present invention may include the step of separatingthe pluripotential stem and progenitor cells from a population ofmononuclear cells obtained from umbilical cord blood using a magneticcell separator to separate out all cells which contain a CD marker, andthen expanding the cells which do not contain a marker in a growthmedium containing a differentiation agent such as retinoic acid, fetalneuronal cells or a growth factor such as BDNF, GDNF and NGF ormixtures, thereof, among numerous others. Preferably, a mixture ofretinoic acid and at least one growth factor, for example, nerve growthfactor, is used as the differentiation agent. The retinoic acid may be9-cis retinoic acid, all-transretinoic acid and mixtures thereof. Theseparation and incubation (differentiation) steps, may be interchanged.

Alternatively, an enriched cell population of pluripotent stem and/orprogenitor cells may be obtained from a population of mononuclear cellsobtained from umbilical cord blood by subjecting the mononuclearpopulation to an amount of an anti-proliferative agent (such as Ara-C[cytidine arabinoside] or methotrexate, among others) effective toeliminate all or substantially all proliferating cells and then exposingthe remaining non-proliferating cells to a mitogen such as epidermalgrowth factor or other mitogen (including other growth factors) toprovide a population of differentiated cells and quiescent cells(pluripotent stem or progenitor cells) which population is grown inculture medium such that the quiescent cells are concentrated in thecell population to greatly outnumber the differentiated cells. Thepluripotent stem and/or progenitor cells obtained may then be grown in acell medium containing a differentiation agent as generally describedabove in order to change the phenotype of the stem and/or progenitorcells to neuronal and/or glial cells which cells may be used intransplantation procedures directly without further purification.

The umbilical cord blood sample from which the pluripotent stem and/orprogenitor cells are obtained may be fresh umbilical cord blood,reconstituted cryopreserved umbilical cord blood or a fresh orreconstituted cryopreserved mononuclear fraction thereof.

Novel compositions according to the present invention comprise umbilicalcord blood or a mononuclear cellular fraction thereof, in combinationwith an effective amount of at least one neural cell differentiationagent. Neural cell differentiation agents for use in the presentinvention include for example, retinoic acid, fetal or mature neuronalcells including mesencephalic or striatal cells or a growth factor orcytokine such as brain derived neurotrophic factor (BDNF), glial derivedneurotrophic factor (GDNF), glial growth factor (GFF) and nerve growthfactor (NGF) or mixtures, thereof. Additional differentiation agentsinclude, for example, growth factors such as fibroblast growth factor(FGF), transforming growth factors (TGF), ciliary neurotrophic factor(CNTF), bone-morphogenetic proteins (BMP), leukemia inhibitory factor(LIF), glial growth factor (GGF), tumor necrosis factors (TNF),interferon, insulin-like growth factors (IGF), colony stimulatingfactors (CSF), KIT receptor stem cell factor (KIT-SCF), interferon,triiodothyronine, thyroxine, erythropoietin, thrombopoietin, silencers,(including glial-cell missing, neuron restrictive silencer factor),antioxidants such as vitamin E (tocopherol) and vitamin E esters, amongothers including lipoic acid, SHC (SRC-homology-2-domain-containingtransforming protein), neuroproteins, proteoglycans, glycoproteins,neural adhesion molecules, and other cell-signaling molecules andmixtures, thereof.

Also presented is a cell line of pluripotent stem and/or progenitorcells produced by any one or more of the above-described methods suchthat the cells have the ability to migrate and localize to specificneuroanatomical regions where they differentiate into neuronal or glialcells typical of the region at the site of transplantation and integrateinto the tissue in a characteristic tissue pattern. Pharmaceuticalcompositions utilizing these cells or other neural cells are also anaspect of the present invention.

The present invention also is directed to a kit for neuronaltransplantation comprising a flask with dehydrated culture medium and apluripotent stem and/or progenitor cells and/or other neural cells.

The present invention is also directed to a method for treating aneurodegenerative (preferably, transplanting in) a patient sufferingfrom such injury, a neurodegenerative disorder or neurological deficitan effective amount of neural and/or neuronal and/or glial cellsaccording to the present invention. Neurodegenerative disorders whichcan be treated using the method according to the present inventioninclude, for example, Parkinson's disease, Huntington's disease,multiple sclerosis (MS), Alzheimer's disease, Tay Sach's disease (betahexosaminidase deficiency), lysosomal storage disease, brain and/orspinal cord injury occurring due to ischemia, spinal cord and braindamage/injury, ataxia and alcoholism, among others, including a numberwhich are otherwise described herein.

The present invention is also directed to a method of treatingneurological damage in the brain or spinal cord which occurs as aconsequence of genetic defect, physical injury, environmental insult ordamage from a stroke, heart attack or cardiovascular disease (most oftendue to ischemia) in a patient, the method comprising administering(including transplanting), an effective number or amount of neural cellsobtained from umbilical cord blood to said patient, including directlyinto the affected tissue of the patient's brain or spinal cord.Administering cells according to the present invention to a patient andallowing the cells to migrate to the appropriate cite within the centralnervous system is another aspect of the present invention.

A method of obtaining neural and/or neuronal and/or glial cells forautologous transplantation from an individual's own umbilical cord bloodcomprises the steps of 1) harvesting mononuclear cells from fresh orcryopreserved umbilical cord blood or a cryopreserved mononuclearfraction of umbilical cord blood; 2) separating out the pluripotent stemcells and/or progenitor cells from the cord blood or mononuclearfraction; 3) incubating the stem cells and/or progenitor cells in amedium which includes an effective amount of a mitogen; and 4)incubating the stem and/or progenitor cells obtained from step 3 with adifferentiation agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C is representative of results of gel studies indicating thepresence of mRNA from neuronal phenotypes.

FIG. 2A-E is representative of microscopic examination of immunostainedcultures of cells which are tested for immunoreactivity with antibodiesto neuronal markers. Certain of the figures evidence that the cells wereimmunoreactive with Mushashi-1 (FIG. 2A), β-tubulin III (FIG. 2B) andGFAP, a marker of astrocytes, (FIG. 2E).

FIGS. 3A, 3B and 3C show the results of neurological function recoveryin animals receiving a mononuclear fraction of human umbilical cordblood 1 day after MCAo as evidenced by adhesive removal, rotarod and NSStests.

FIGS. 4A, 4B and 4C show the results of neurological function recoveryin animals receiving a mononuclear fraction of human umbilical cordblood 7 days after MCAo as evidenced by adhesive removal, rotarod andNSS tests.

FIG. 5 shows the results of immunostaining of brain sections withMAB1281 and evidences that the highest concentrations of cord bloodcells migrate to the injured tissue.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following definitions are used throughout the specification todescribe the present invention.

The term “patient” is used throughout the specification to describe ananimal, preferably a human, to whom treatment, including prophylactictreatment, with the compositions according to the present invention, isprovided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patient,the term patient refers to that specific animal. The term “donor” isused to describe an individual (animal, including a human) who or whichdonates umbilical cord blood for use in a patient.

The term “umbilical cord blood” or “cord blood” is used throughout thespecification to refer to blood obtained from a neonate or fetus, mostpreferably a neonate and preferably refers to blood which is obtainedfrom the umbilical cord or the placenta of newborns. The use of cord orplacental blood as a source of mononuclear cells is advantageous becauseit can be obtained relatively easily and without trauma to the donor. Incontrast, the collection of bone marrow cells from a donor is atraumatic experience. Cord blood cells can be used for autologoustransplantation or allogenic transplantation, when and if needed. Cordblood is preferably obtained by direct drainage from the cord and/or byneedle aspiration from the delivered placenta at the root and atdistended veins.

The term “effective amount” is used throughout the specification todescribe concentrations or amounts of components such as differentiationagents, mitogens, neural and/or neuronal or glial cells, or other agentswhich are effective for producing an intended result includingdifferentiating stem and/or progenitor cells into neural, neuronaland/or glial cells, or treating a neurodegenerative disease or otherneurological condition including damage to the central nervous system ofa patient, such as a stroke, heart attack or accident victim or foreffecting a transplantation of those cells within the patient to betreated. Compositions according to the present invention may be used toaffect a transplantation of the neural cells within the composition toproduce a favorable change in the brain or spinal cord, or in thedisease or condition treated, whether that change is an improvement(such as stopping or reversing the degeneration of a disease orcondition, reducing a neurological deficit or improving a neurologicalresponse) or a complete cure of the disease or condition treated.

The term “neural cells” are cells having at least an indication ofneuronal or glial phenotype, such as staining for one or more neuronalor glial markers or which will differentiate into cells exhibitingneuronal or glial markers. Examples of neuronal markers which may beused to identify neuronal cells according to the present inventioninclude, for example, neuron-specific nuclear protein, tyrosinehydroxylase, microtubule associated protein, and calbindin, amongothers. The term neural cells also includes cells which are neuralprecursor cells, i.e., stem and/or progenitor cells which willdifferentiate into or become neural cells or cells which will ultimatelyexhibit neuronal or glial markers, such term including pluripotent stemand/or progenitor cells which ultimately differentiate into neuronaland/or glial cells. All of the above cells and their progeny areconstrued as neural cells for the purpose of the present invention.Neural stem cells are cells with the ability to proliferate, exhibitself-maintenance or renewal over the life-time of the organism and togenerate clonally related neural progeny. Neural stem cells give rise toneurons, astrocytes and oligodendrocytes during development and canreplace a number of neural cells in the adult brain. Neural stem cellsare neural cells for purposes of the present invention. The terms“neural cells” and “neuronal cells” are generally used interchangeablyin many aspects of the present invention. Preferred neural cells for usein certain aspects according to the present invention include thosecells which exhibit one or more of the neural/neuronal phenotypicmarkers such as Mushashi-1, Nestin, NeuN, class III β-tubulin, GFAP,NF-L, NF-M, microtubule associated protein (MAP2), S1100, CNPase,glypican (especially glypican 4), neuronal pentraxin II, neuronal PAS 1,neuronal growth associated protein 43, neurite outgrowth extensionprotein, vimentin, Hu, intemexin, 04, myelin basic protein andpleiotrophin, among others.

The term “administration” or “administering” is used throughout thespecification to describe the process by which neural cells according tothe present invention are delivered to a patient for treatment purposes.Neural cells may be administered a number of ways including parenteral(such term referring to intravenous and intraarterial as well as otherappropriate parenteral routes), intrathecal, intraventricular,intraparenchymal (including into the spinal cord, brainstem or motorcortex), intracisternal, intracranial, intrastriatal, and intranigral,among others which term allows neural cells to migrate to the cite whereneeded. Neural cells may be administered in the form of whole cord bloodor a fraction thereof (such term including a mononuclear fractionthereof or a fraction of neural cells, including a high concentration ofneural cells). The compositions according to the present invention maybe used without treatment with a differentiation agent (“untreated”,i.e., without further treatment in order to promote differentiation ofcells within the umbilical cord blood sample) or after treatment(“treated”) with a differentiation agent or other agent which causescertain pluripotential stem and/or progenitor cells within the cordblood sample to differentiate into cells exhibiting neuronal and/orglial phenotype. Administration will often depend upon the disease orcondition treated and may preferably be via a parenteral route, forexample, intravenously, by administration into the cerebral spinal fluidor by direct administration into the affected tissue in the brain. Forexample, in the case of Alzheimer's disease, Huntington's disease andParkinson's disease, the preferred route of administration will be atransplant directly into the striatum (caudate cutamen) or directly intothe substantia nigra (Parkinson's disease). In the case of amyotrophiclateral sclerosis (Lou Gehrig's disease) and multiple sclerosis, thepreferred administration is through the cerebrospinal fluid. In the caseof lysosomal storage disease, the preferred route of administration isvia an intravenous route or through the cerebrospinal fluid. In the caseof stroke, the preferred route of administration will depend upon wherethe stroke is, but will often be directly into the affected tissue(which may be readily determined using MRI or other imaging techniques).

Each of these conditions, however, may be readily treated using otherroutes of administration including, for example, an intravenous orintraarterial administration of whole umbilical cord blood or amononuclear cell fraction thereof to treat a condition or disease state.In the case of such treatment, however, and in particular, the treatmentof amyotrophic lateral sclerosis (Lou Gehrig's disease), treatment ofthe patient using parenteral (in particular, intravenous orintraarterial) administration of whole umbilical cord blood or amononuclear cellular fraction thereof or other routes of administrationwill be performed preferably in the absence of radiation or othertreatment such as chemotherapy (which are often used to eliminate bonemarrow cells or other tissue in the patient in order to impair, destroyand replace hematopoietic cells) before, during or after administrationof the umbilical cord blood or mononuclear cell fraction, thereof.

The terms “grafting” and “transplanting” and “graft” and“transplantation” are used throughout the specification synonymously todescribe the process by which neural and/or neuronal cells according tothe present invention are delivered to the site within the nervoussystem where the cells are intended to exhibit a favorable effect, suchas repairing damage to a patient's central nervous system, treating aneurodegenerative disease or treating the effects of nerve damage causedby stroke, cardiovascular disease, a heart attack or physical injury ortrauma or genetic damage or environmental insult to the brain and/orspinal cord, caused by, for example, an accident or other activity.Neural cells for use in the present invention may also be delivered in aremote area of the body by any mode of administration as describedabove, relying on cellular migration to the appropriate area in thecentral nervous system to effect transplantation.

The term “essentially” is used to describe a population of cells or amethod which is at least 95+% effective, more preferably at least about98% effective and even more preferably at least 99% effective. Thus, amethod which “essentially” eliminates a given cell population,eliminates at least about 95+% of the targeted cell population, mostpreferably at least about 99% of the cell population. Neural cellsaccording to the present invention, in certain preferred embodiments,are essentially free of hematopoietic cells (i.e., the CD34+ cellularcomponent of the mononuclear cell fragment).

The term “non-tumorigenic” refers to the fact that the cells do not giverise to a neoplasm or tumor. Stem and/or progenitor cells for use in thepresent invention are generally free from neoplasia and cancer.

The term “cell medium” or “cell media” is used to describe a cellulargrowth medium in which mononuclear cells and/or neural cells are grown.Cellular media are well known in the art and comprise at least a minimumessential medium plus optional agents such as growth factors, glucose,non-essential amino acids, insulin, transferrin and other agents wellknown in the art. In certain preferred embodiments, at least onedifferentiation agent is added to the cell media in which a mononuclearcell fraction is grown in order to promote differentiation of certaincells within the mononuclear fraction into neural cells.

In a preferred aspect of the present invention, mononuclear cells grownin standard cellular media (preferably, at least a minimum essentialmedium supplemented with non-essential amino acids, glutamine,mercaptoethanol and fetal bovine serum (FBS)) are grown in a “neuralproliferation medium” (i.e., a medium which efficiently grows neuralcells) followed by growth in a “differentiation medium”, generally,which is similar to the neural proliferation medium with the exceptionthat specific nerve differentiation agents are added to the medium andin certain cases, other growth factors are limited or removed). Aparticularly preferred neural proliferation medium is a media whichcontains DMEM/F12 1:1 cell media, supplemented with 0.6% glucose,insulin (25 μg/ml), transferrin (100 μg/ml), progesterone 20 nM,putrescine (60 μM, selenium chloride 30 nM, glutamine 2 mM, sodiumbicarbonate 3 mM, HEPES 5 mM, heparin 2 μg/ml and EGF 20 nm/ml, bFGF 20ng/ml. One of ordinary skill will readily recognize that any number ofcellular media may be used to grow mononuclear cell fractions ofumbilical cord blood or to provide appropriate neural proliferationmedia and/or differentiation media.

The term “separation” is used throughout the specification to describethe process by which pluripotent stem and/or progenitor cells areisolated from a mononuclear cell sample or a sample which contains cellsother than the desirable stem and/or progenitor cells, for example,umbilical cord blood or other fragment.

The term “mitogen” is used throughout the specification to describe anagent which is added to non-proliferating cells obtained from amononuclear cell sample in order to produce differentiated cells andquiescent cells (pluripotent stem and/or progenitor cells). A mitogen isa transforming agent which induces mitosis in certain cells other thanpluripotent stem and/or progenitor cells obtained from umbilical cordblood. Preferred mitogens for use in the present invention includeepidermal growth factor (EGF), among other agents such as the lesspreferred pokeweed mitogen, which also may be used to induce mitosis.Mitogens are also any one or a combination of a variety of growthfactors which have been shown to exert mitogenic actions on neural andmesenchymal precursors. These growth factors are: Epidermal GrowthFactor (EGF) family ligands (EGF, Transforming Growth Factor α,amphiregulin, betacellulin, heparin-binding EGF and Heregulin), basicFibroblastic growth factors (bFGF) and other members of its super-family(FGF1, FGF4), members of Platelet-Derived Growth Factor family (PDGF AA,AB, BB), Interleukins, and members of the Transforming Growth Factor βsuperfamily.

The term “antiproliferative agent” is sued throughout the specificationto describe an agent which will prevent the proliferation of cellsduring methods according to the present invention which enrichpluripotent stem and/or progenitor cells. Exemplary antiproliferativeagents include, for example, Ara-C, methotrexate and otherantiproliferative agents. Preferred antiproliferative agents are thoseagents which limit or prevent the growth of proliferating cells withinan umbilical cord blood sample or mononuclear cell fraction thereof sothat quiescent stem and/or progenitor cells may be enriched.

The term “differentiation agent” or “neural differentiation agent” isused throughout the specification to describe agents which may be addedto cell culture (which term includes any cell culture medium which maybe used to grow neural cells according to the present invention)containing pluripotent stem and/or progenitor cells which will inducethe cells to a neuronal or glial phenotype. Preferred differentiationagents for use in the present invention include, for example,antioxidants, including retinoic acid, fetal or mature neuronal cellsincluding mesencephalic or striatal cells or a growth factor or cytokinesuch as brain derived neurotrophic factor (BDNF), glial derivedneurotrophic factor (GDNF) and nerve growth factor (NGF) or mixtures,thereof. Additional differentiation agents include, for example, growthfactors such as fibroblast growth factor (FGF), transforming growthfactors (TGF), ciliary neurotrophic factor (CNTF), bone-morphogeneticproteins (BMP), leukemia inhibitory factor (LIF), glial growth factor(GGF), tumor necrosis factors (TNF), interferon, insulin-like growthfactors (IGF), colony stimulating factors (CSF), KIT receptor stem cellfactor (KIT-SCF), interferon, triiodothyronine, thyroxine,erythropoietin, thrombopoietin, silencers, (including glial-cellmissing, neuron restrictive silencer factor), SHC(SRC-homology-2-domain-containing transforming protein), neuroproteins,proteoglycans, glycoproteins, neural adhesion molecules, and othercell-signaling molecules and mixtures, thereof. Differentiation agentswhich can be used in the present invention are detailed in“Marrow-mindedness: a perspective on neuropoiesis”, by Bjorn Scheffler,et al., TINS, 22, pp. 348-356 (1999), which is incorporated by referenceherein.

The term “neurodegenerative disease” is used throughout thespecification to describe a disease which is caused by damage to thecentral nervous system and which damage can be reduced and/or alleviatedthrough transplantation of neural cells according to the presentinvention to damaged areas of the brain and/or spinal cord of thepatient. Exemplary neurodegenerative diseases which may be treated usingthe neural cells and methods according to the present invention includefor example, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis (Lou Gehrig's disease), Alzheimer's disease, RettSyndrome, lysosomal storage disease (“white matter disease” orglial/demyelination disease, as described, for example by Folkerth, J.Neuropath. Exp. Neuro., 58, 9, September, 1999), including Sanfilippo,Tay Sachs disease (beta hexosaminidase deficiency), other geneticdiseases, multiple sclerosis, brain injury or trauma caused by ischemia,accidents, environmental insult, etc., spinal cord damage, ataxia andalcoholism. In addition, the present invention may be used to reduceand/or eliminate the effects on the central nervous system of a strokeor a heart attack in a patient, which is otherwise caused by lack ofblood flow or ischemia to a site in the brain of said patient or whichhas occurred from physical injury to the brain and/or spinal cord. Theterm neurodegenerative disease also includes neurodevelopmentaldisorders including for example, autism and related neurologicaldiseases such as schizophrenia, among numerous others.

Selecting for umbilical cord pluripotential stem and/or progenitor cellsaccording to the present invention can be done in a number of ways. Forexample, the cells may be selected using, for example a magnetic cellseparator (FACS) or other system which removes all cells which contain aCD marker and then the remaining cells may be expanded in growth mediumor differentiated in growth medium which includes a differentiationagent. Alternatively, an enriched population of stem and/or progenitorcells may be obtained from a sample of mononuclear cells by subjectingthe cells to an agent such as Ara-C or other anti-proliferative agentsuch as methotrexate, which causes the death of proliferating cellswithin a sample (the stem and/or progenitor cells are non-proliferatingand are unaffected by the agent). The remaining cells may then be grownin a cell culture medium which contains a mitogen to produce apopulation of differentiated and quiescent cells, which cell populationmay be further grown to concentrate the quiescent cells to the effectiveexclusion of the differentiated cells (the quiescent cells in the finalcell medium will greatly outnumber the original differentiated cellswhich do not grow in the medium). The quiescent cells may then beinduced to adopt a number of different neural phenotypes, which cellsmay be used directly in transplantation.

Additional in vitro differentiation techniques can be adapted throughthe use of various cell growth factors and co-culturing techniques knownin the art. Besides co-culturing with fetal mesencephalic or striatalcells, a variety of other cells can be used, including but not limitedto accessory cells, and cells from other portions of the fetal andmature central nervous system.

The term “gene therapy” is used throughout the specification to describethe transfer and stable insertion of new genetic information into cellsfor the therapeutic treatment of diseases or disorders. The foreign geneis transferred into a cell that proliferates to spread the new genethroughout the cell population. Thus, stem cells, or pluripotentprogenitor cells according to the present invention either prior todifferentiation or preferably, after differentiation to a neural cellphenotype, are the target of gene transfer, since they are proliferativecells that produce various progeny lineages which will potentiallyexpress the foreign gene.

The following written description provides exemplary methodology andguidance for carrying out many of the varying aspects of the presentinvention.

General Methods

Standard molecular biology techniques known in the art and notspecifically described are generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory,New York (1989, 1992), and in Ausubel et al., Current Protocols InMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).Polymerase chain reaction (PCR) is carried out generally as in PCRProtocols: A Guide To Methods and Applications, Academic Press, SanDiego, Calif. (1990). Reactions and manipulations involving othernucleic acid techniques, unless stated otherwise, are performed asgenerally described in Sambrook, et al., 1989, Molecular Cloning: aLaboratory Manual, Cold Springs Harbor Laboratory Press, and methodologyas set forth in U.S. Pat. Nos. 4,666,828; 4,683,202, 4,801,531;5,192,659; and 5,272,057 and incorporated herein by reference. In-situPCR in combination with Flow Cytometry can be used for detection ofcells containing specific DNA and mRNA sequences (see, for example,Testoni et al. Blood 87:3822 (1996)).

Standard methods in immunology known in the art and not specificallydescribed are generally followed as in Stites et al. (eds), BASIC ANDCLINICAL IMMUNOLOGY, 8th Ed., Appleton & Lange, Norwalk, Conn. (1994);and Mishell and Shigi (eds), Selected Methods In Cellular Immunology,W.H. Freeman and Co., New York (1980).

Immunoassays

In general, immunoassays are employed to assess a specimen such as forcell surface markers or the like. Immunocytochemical assays are wellknown to those skilled in the art. Both polyclonal and monoclonalantibodies can be used in the assays. Where appropriate otherimmunoassays, such as enzyme-linked immunosorbent assays (ELISAs) andradioimmunoassays (RIA), can be used as are known to those in the art.Available example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752;3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074;3,984,533; 3,996,345; 4,034,074; 4,098,876; 2o 4,879,219; 5,011,771 and5,281,521 as well as Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor, N.Y. (1989). Numerous other references alsomay be relied on for these teachings.

Antibody Production

Antibodies may be monoclonal, polyclonal or recombinant. Conveniently,the antibodies may be prepared against the immunogen or immunogenicportion thereof, for example, a synthetic peptide based on the sequence,or prepared recombinantly by cloning techniques or the natural geneproduct and/or portions thereof may be isolated and used as theimmunogen. Immunogens can be used to produce antibodies by standardantibody production technology well known to those skilled in the art asdescribed generally in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Springs Harbor, N.Y. (1988) andBorrebaeck, Antibody Engineering—A Practical Guide by W. H. Freeman andCo. (1992). Antibody fragments may also be prepared from the antibodiesand include Fab and F(ab′)2 by methods known to those skilled in theart. For producing polyclonal antibodies, a host, such as a rabbit orgoat, is immunized with the immunogen or immunogenic fragment, generallywith an adjuvant and, if necessary, coupled to a carrier; antibodies tothe immunogen are collected from the serum. Further, the polyclonalantibody can be absorbed such that it is monospecific. That is, theserum can be exposed to related immunogens so that cross-reactiveantibodies are removed from the serum rendering it monospecific.

For producing monoclonal antibodies, an appropriate donor ishyperimmunized with the immunogen, generally a mouse, and splenicantibody-producing cells are isolated. These cells are fused to immortalcells, such as myeloma cells, to provide a fused cell hybrid that isimmortal and secretes the required antibody. The cells are thencultured, and the monoclonal antibodies harvested from the culturemedia.

For producing recombinant antibodies, messenger RNA fromantibody-producing B-lymphocytes of animals or hybridoma isreverse-transcribed to obtain complementary DNAs (cDNAs). Antibody cDNA,which can be full or partial length, is amplified and cloned into aphage or a plasmid. The cDNA can be a partial length of heavy and lightchain cDNA, separated or connected by a linker. The antibody, orantibody fragment, is expressed using a suitable expression system.Antibody cDNA can also be obtained by screening pertinent expressionlibraries. The antibody can be bound to a solid support substrate orconjugated with a detectable moiety or be both bound and conjugated asis well known in the art. (For a general discussion of conjugation offluorescent or enzymatic moieties see Johnstone & Thorpe,Immunochemistry in Practice, Blackwell Scientific Publications, Oxford,1982). The binding of antibodies to a solid support substrate is alsowell known in the art. (see for a general discussion Harlow & Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPublications, New York, 1988 and Borrebaeck, Antibody Engineering—APractical Guide, W.H. Freeman and Co., 1992). The detectable moietiescontemplated with the present invention can include, but are not limitedto, fluorescent, metallic, enzymatic and radioactive markers. Examplesinclude biotin, gold, ferritin, alkaline phosphates, galactosidase,peroxidase, urease, fluorescein, rhodamine, tritium, ¹⁴C, iodination andgreen fluorescent protein.

Gene Therapy

Gene therapy as used herein refers to the transfer of genetic material(e.g., DNA or RNA) of interest into a host to treat or prevent a geneticor acquired disease or condition. The genetic material of interestencodes a product (e.g., a protein. polypeptide. and peptide, functionalRNA, antisense) whose in vivo production is desired. For example, thegenetic material of interest encodes a hormone, receptor, enzyme,polypeptide or peptide of therapeutic value. Alternatively, the geneticmaterial of interest encodes a suicide gene. For a review see “GeneTherapy” in Advances In Pharmacology, Academic Press, San Diego, Calif.,1997.

Administration of Cells for Transplantation

The cells of the present invention are administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement, including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the cells of the presentinvention can be administered in various ways as would be appropriate toimplant in the central nervous system, including but not limited toparenteral, including intravenous and intraarterial administration,intrathecal administration, intraventricular administration,intraparenchymal, intracranial, intracisternal, intrastriatal, andintranigral administration. In addition, all of these routes ofadministration may be used to effect transplantation of neural cells inthe present invention.

Methods of treating a patient for a neurodegenerative disease or brainand/or spinal cord damage caused by, for example, physical injury or byischemia caused by, a stroke, heart attack or cardiovascular diseasecomprise administering neural cells to said patient in an amountsufficient to affect a neuronal transplantation. One of ordinary skillmay readily recognize that one may use treated (i.e., cells exposed toat least one differentiation agent) or untreated neural cells for suchmethods, including fresh umbilical cord blood or a mononuclear fractionthereof.

Pharmaceutical compositions comprising effective amounts of treatedneural cells are also contemplated by the present invention. Thesecompositions comprise an effective number of treated neural and/orneuronal and/or glial cells, optionally in combination with apharmaceutically acceptable carrier, additive or excipient. In certainaspects of the present invention, cells are administered to the patientin need of a transplant in sterile saline. In other aspects of thepresent invention, the cells are administered in Hanks Balanced SaltSolution (HBSS) or Isolyte S, pH 7.4. Other approaches may also be used,including the use of serum free cellular media. Such compositions,therefore, comprise effective amounts or numbers of treated neural cellsin sterile saline. These may be obtained directly by using fresh orcryopreserved umbilical cord blood or alternatively, by separating outthe mononuclear cells (MNC) from the whole blood, using density gradientseparation methods, among others, which are well known in the art (onesuch approach is presented herein). The isolated MNC may be useddirectly for administration/transplantation or may be treated with atleast one differentiation agent and used without further purification orisolation of neural cells, or alternatively, after treatment with atleast one differentiation agent, the neural cells may be isolated andused. Intravenous or intraarterial administration of the cells insterile saline to the patient may be preferred in certain indications,whereas direct administration at the site of or in proximity to thediseased and/or damaged tissue may be preferred in other indications.

Pharmaceutical compositions according to the present inventionpreferably comprise an effective number within the range of about 1×10⁴mononuclear cells to about 5×10⁷ mononuclear cells, more preferablyabout 1×10⁵ to about 9×10⁶ mononuclear cells, even more preferably about1×10⁶ to about 8×10⁶ cells generally in solution, optionally incombination with a pharmaceutically acceptable carrier, additive orexcipient. Effective numbers of neural cells, either within a sample ofmononuclear cells or as concentrated or isolated neural cells, may rangefrom as few as several hundred or fewer to several million or more,preferably at least about one thousand cells within this range. Inaspects of the present invention whereby the cells are injected inproximity to the brain or spinal cord tissue to be treated, the numberof cells may be reduced as compared to aspects of the present inventionwhich rely on parenteral administration (including intravenous and/orintraarterial administration).

In using compositions according to the present invention, fresh orcryopreserved umbilical cord blood, a mononuclear fraction thereof, orfractions wherein neural cells are isolated and/or concentrated (usingFACS or other separation methods for isolating neural cells from apopulation of mononuclear cells) may be used without treatment with adifferentiation agent or with an effective amount of a differentiationagent prior to being used in a neuronal transplant. In one preferredaspect of the present invention, a mononuclear fraction of cord blood isexposed to effective amounts of at least one differentiation agent (incell media) for a period to effect differentiation of cord stem cellsinto cells which express a neuronal and/or glial phenotype and aftersuch period, these cells are then used to affect a transplant in apatient.

In aspects of the invention in which cord blood stem cells aredifferentiated, the use of standard media which has been supplementedwith at least one or more differentiation agent is preferred. Acombination of retinoic acid and nerve growth factor (NGF) in effectiveamounts in certain aspects of the present invention as thedifferentiation agent is preferred. In certain preferred aspects of thepresent invention, neural cells are prepared from cord blood stems cellsgrowing in standard growth media in a two-step approach using neuralproliferation media followed by a differentiation media. In this aspectof the present invention, cells grown in standard cellular media(preferably, at least a minimum essential medium supplemented withnon-essential amino acids, glutamine, mercaptoethanol and fetal bovineserum (FBS)) are initially grown in a “neural proliferation medium”(i.e., a medium which efficiently grows neural cells) followed by growthin a “differentiation medium”) (generally, similar to the neuralproliferation medium with the exception that specific nervedifferentiation agents are added to the medium and in certain cases,other growth factors are limited or removed). A preferred neuralproliferation medium is a media which contains DMEM/F12 1:1 cell media,supplemented with 0.6% glucose, insulin (25 μg/ml), transferrin (100μg/ml), progesterone 20 nM, putrescine (60 μM, selenium chloride 30 nM,glutamine 2 mM, sodium bicarbonate 3 mM, HEPES 5 mM, heparin 2 μg/ml andEGF 20 nm/ml, bFGF 20 ng/ml. One of ordinary skill will readilyrecognize that any number of cellular media may be used to provideappropriate neural proliferation medium and/or differentiation medium.

The following examples are provided to illustrate or exemplify certainpreferred embodiments of the present invention illustrative of thepresent invention but are not intended in any way to limit the presentinvention.

Examples

Preparation of Cellular Samples

Cryopreserved or fresh umbilical cord blood (from human or rat umbilicalcord that remains attached to placenta after delivery) is harvested andprocessed by Ficoll centrifugation. This results in nearly 100% recoveryof mononuclear cells which can be a) grafted directly into a region ofinjured brain (e.g. in a rat stroke model or model of neurodegenerativedisease or trauma model) b) processed into sub-populations based onsurface markers or c) cryopreserved for later use. Initial experimentswith umbilical cord blood utilize all of the mononuclear cellscollected, without separation of CD34+ cellular components(hematopoietic stem cells). Other experiments utilize cord blood that isdepleted of CD34+ cells as described below. Approximately 100,000 to90,000,000 (1×10⁵ to about 9×10⁷, preferably at least about 1×10⁶) cordblood cells are injected into the hemisphere rendered ischemic byacutely obstructing blood flow to cerebral cortex. Assessment ofrecovery of limb function in the rat model of stroke is performed at 2and 4 and 8 weeks after grafting.

Preparation of Cord Blood Devoid of Hematopoietic Stem Cells (CD34+)

Using a magnetic cell sorting kit (Milteny Biotec, Inc, Auburn Tx), cordcells are labeled with CD34+ microbeads which marks cells that expresshematopoietic stem cell antigen (CD34 in human samples). The cord bloodcells are passed through an MS+ column for selection of CD34+ cells. Twohundred μL of CD34+ Multi-sort MicroBeads is added per 10⁸ total cells,mixed and incubated for 15 min at 6-12° C. Cells are washed by adding5-10× the labeling volume of buffer, centrifuged for 10 min at 200×g andsupernatant is removed. The cell pellet is resuspended in 500 μL buffer.The MS+/RS+ column is washed with 500 μL of buffer. The cell suspensionis applied to the column and the “negative” cells passed through. Thenegative cells contain all cells in the cord blood except thehematopoietic CD34+ cells. The column is then rinsed with 500 μL ofbuffer three times. These washing are added to the “negative” cellfraction, centrifuged for 10 min at 220×g and the supernatant removed.The cell pellet is resuspended in 500 μL of buffer and diluted 1:1 withDulbecco's Minimal Essential Media (DMEM, GIBCO/BRL) and 10% fetalbovine serum (FBS), centrifuged through a density gradient (Ficoll-PaquePlus, 1.077 g/ml, Pharmacia) for 30 min at 1,000×g. The supernatant andinterface are combined and diluted to approximately 20 ml with growthmedium and plated in polyethylene-imine coated plastic flasks.

Defining the Optimal Medium for Generating Cord Blood Clones

The cord blood is suspended in serum-free medium composed of a 1:1mixture of Dulbecco's Minimal Essential Media (DMEM) and F12 nutrient(Life Technologies-BRL). Other samples of cord blood material aresuspended in DMEM+10% Fetal Bovine Serum (FBS). The defined culturemedium is composed of DMEM/F12 (1:1) including glucose, glutamine,sodium bicarbonate and HEPES buffer plus a defined hormone and saltmixture (see Daadi and Weiss, 1999). To identify the optimal celldensity for cell survival and growth, cells are plated at densitiesvarying from 50,000 to 3×10⁶ cell/ml in Corning T75 culture flasks inthe defined media together with a specific mitogenic growth factor(s)(see below for the mitogens used). Cell survival and proliferation aremonitored very closely by examining culture flasks, noting and countingclones that arise daily. After a fixed culture period of 10 days thetotal number of clones are counted in both serum free and serumsupplemented cultures and compared. This gave us the number of precursorcells able to generate clones and rate of proliferation. Then theseclones are harvested, dissociated and the total viable cells counted andreseeded for a second passage and so on for the future passages. Thislast count allowed determination of the rate of proliferation and thesize of clones generated under each condition.

Caveats:

From experience with neural stem cells, the inventors have found thatexposure of cells to serum induces differentiation and inhibitsproliferation of neural lineages. Lower cell density also does not favorcell proliferation. Therefore, it is likely that the inventors willobserve variability in the rate of cell growth depending on the presenceand absence of serum, the cell density and mitogen used. The mediacomposition may not be optimal for cell survival, proliferation andenrichment for a neural cell population. The inventors also seedifferences in morphologies and antigens expressed by the cells underthese two separate conditions. Therefore, before each passage theinventors identify the total number of clones and cells and also theproportion of neurons, astrocytes and oligodendrocytes as well ashematopoietic cell lineages. These data will guide us to constantlyimprove the medium formula by trying new media components and, ifnecessary, a very low percentage of Bovine Serum Albumin (BSA) or neuralstem cell conditioned media.

Identification of Sub-Populations of Cord Blood Cells Responsive toSpecific Mitogens and which Express Specific Neural Markers

The cord blood suspension is plated at an optimal cell density in comingT75 culture flasks in the optimal medium (as described above). Thismedium is supplemented with 10 to 20 ng/ml of one or a combination of avariety of growth factors that have been shown to exert mitogenicactions on neural and mesenchymal precursors. These growth factors are:Epidermal Growth Factor (EGF) family ligands (EGF, Transforming GrowthFactor α, amphiregulin, betacellulin, heparin-binding EGF andHeregulin), basic Fibroblastic growth factors (bFGF) and other membersof its super-family (FGF1, FGF4), members of Platelet-Derived GrowthFactor family (PDGF AA, AB, BB), Interleukins, and members of theTransforming Growth Factor β superfamily. After a period of 10 to 15days in vitro, the cells are harvested and then reseeded in fresh mediumcontaining growth factor(s). To identify their immature nature, some ofthese cells are plated on poly-L-ornithine-coated glass coverslips in24-well Nunclon culture dishes. After, a period of 30 minutes to 1 hourthese cells are fixed with 4% paraformaldehyde and stained with avariety of markers for immature cells such as Nestin, vimentin, the CDmarkers CD34 (marker of hematopoietic stem cell) and CD33 and dendriticcell markers. The rest of the cells are reseeded in growth medium(medium containing mitogens) for the next passage. For each growthfactor used cell survival and proliferation is closely monitored andclones that arise every day are counted and the total number of clonesformed after a fixed period is determined. The inventors also determinethe proportion of cell types generated under each mitogen, as describedbelow.

Different rates of cell proliferation and/or proportion of cell typesare generated depending on the growth factor used and its concentration.Neuronal differentiation efficacy and the number of passages able to becarried out under each mitogen is determined. Studies have shown thatthe combination of EGF and FGF is required to isolate and propagatehuman neural stem cell. Therefore, the inventors test combinations ofthese growth factors and potentiated the mitogenic action by adding aspecific component (heparin when bFGF is used as the mitogen).

Establishment of Multipotent Clonally Derived Sub-Populations of CordBlood Stem Cells.

Isolation of precursor sub-populations based on their response toepigenetic signals generate a homogeneous cell population that behave ina predictable and similar manner when transplanted in vivo or challengedwith a specific treatment in vitro. These clonal cell lines are goodcandidates for clinical-grade development. From the initial startinggrowth factor responsive population of stable human cord blood cells,monoclonal cell populations are established as previously described(Daadi and Weiss, J. Neurosci. 19, 11 4484, 1999). Clusters of cells aredissociated, counted and suspended in media-hormone mix at aconcentration of 1 cell per 15 μl and plated at 15 μl/well in Terasakimicrowells or a 96-well dish. Wells with single cells are immediatelyidentified and marked. Single cells are also randomly picked from thesuspension using a hand-pulled 10 μl micropipette and transferred into aTerasaki microwell containing 10-15 μl of media. Clonal development ismonitored once per day using the inverted microscope with phase-contrastoptics. Cultures are fed by replacing 2 μl with fresh medium every 2days.

Each single cell proliferates and generates a clone of cells. A fractionof these single founder cells have a slow growth rate or do notproliferate or even die after a few days in culture. From experiencewith neural stem cells and using clonal cultures, some single cells mayundergo cell death because of the lack of neighboring cells that provideextracellular support for cell survival and in specific cases celldivision. If this is a problem, the founder cells are cultured inconditioned medium derived from bulk stem cell cultures, or in thepresence of the membrane extract of cord blood cells.

Characterization and Determination of the Differentiation Efficacy ofEach Clone.

In addition to growing a purified monoclonal human cord blood-derivedstem cell populations, it is necessary to verify that each generation ofthe clone exhibits all stem cell characteristics i.e.: ability toself-renew, generate a large number of progeny and be able to respond toenvironmental cues and differentiate into different cell types. Theseefficacy criteria are fundamental for the development and the productionof stable multipotent clones. Clonally derived cells (as describedabove) are dissociated either by gentle mechanical trituration or usingtrypsin-EDTA. After the growth phase, part of the next generation cloneis cultured under differentiation conditions. When cells grow as acluster in suspension each clone is removed and plated in controlmedia-hormone mixture without any mitogens on a glass coverslip coatedwith an extracellular matrix (ECM). Different ECMs including laminin,Poly-L-ornithine and poly-D-lysine are tested for their potentialdifferentiation effects. If cells grow as a monolayer, media containingthe mitogen is removed by gentle suction and replaced by control freshmedia (no mitogen). After a culture period of 10 to 15 days,differentiated cells are fixed with paraformaldehyde and stained forvarious neural and, hematopoietic cell markers. Analysis of labeledsubpopulations are carried out using immunocytochemical techniques andFlow Cytometric Analysis. For neural lineages, the inventors use:anti-Nestin, and anti-Vimentin to label immature precursor cells;anti-Glial Fibrillary Acidic Protein to label Astrocytes, anti-O4,anti-Myelin Basic Protein and anti-CNPase to identify oligodendrocytes,Anti-NeuN, Anti-β-tubulin class III, Anti-Neuron Specific Enolase,Anti-human specific Neurofilament, Anti-MAP2 to identify neurons. Withinthis last neuronal population, the inventors test for differentneurotransmitter phenotype expression like GABA, CholineAcetyltransferase, Tyrosine Hydroxylase and Serotonin. The inventorsalso test for hematopoietic cells: Some of the characterized multipotentstem cell clones are cryopreserved as described in general methodssection (see below) and the rest passaged and maintained in culture.

Cryopreservation

Clonally derived cord blood cells are resuspended in cell freezing mediacomprising 10% dimethyl sulfoxide, 50% Fetal Bovine Serum and 40% ofdefined medium and stored under liquid nitrogen are well known in theart.

The mononuclear layer from whole umbilical cord blood may be preparedfor cryopreservation using the following methodology, which steps may bevaried without significantly changing the cryopreservation outcome.

Processing and Storage of Umbilical Cord Blood

1. Sample Preparation

Anticoagulated cord blood is aliquotted into sterile 50 ml conical tubesand the volume measured accurately. A small sample is removed for whitecell count and sterility testing. A sample of plasma is removed at thistime by centrifugation for cryopreservation. The cord blood is diluted1:2 with sterile phosphate buffered saline (PBS) and mixed carefully toa maximum of 35 ml per tube.

Step 2: Density Gradient Separation

Mononuclear cells are obtained from the cord blood using Ficoll-Hypaquedensity centrifugation. Each tube of diluted cord blood is underlayeredwith 10 ml of sterile Ficoll-Hypaque solution and then centrifuged at1200 g for 30 min. at room temperature. In this procedure, mononuclearcells containing progenitor cells (stem cells) form a layer at theFicoll/plasma interface whereas red cells and granular cells(granulocytes) pass through the gradient to the bottom of the tube. Themononuclear cells are removed carefully by aspiration.

Step 3: Mononuclear Cell Preparation (MNC)

The mononuclear cells are collected in sterile 50 ml tubes and diluted1:2 with tissue culture medium (RPMI) and centrifuged at 1500 g for 15minutes. The cells are further washed in RPMI and resuspended to a fixedvolume (14 ml) and a small sample removed for white cell enumeration andCD34+ cell determination.

Step 4: Preparation of MNC for Cryopreservation

The cell suspension is then centrifuged at 1200 g for 10 mins and thecells resuspended in 2.5 ml of RPMI. A small sample is removed forsterility testing. To this suspension. 2.5 ml of autologous plasmacontaining 10% dimethyl sulfoxide (DMSO) as cryoprotectant is addedslowly and the resulting suspension transferred to a labeled (bar coded)sterile, 5 ml cryovial.

Step 5: Controlled Rate Freezing in Liquid Nitrogen

The samples are then cryopreserved using a controlled rate of freezingfrom 4° C. to −90° C. using the following protocol:

+4° C. to −3° C. at one degree C. per minute

−3° C. to −20° C. at 10 degrees C. per minute

−20° C. to −40° C. at one degree C. per minute

−40° C. to −90° C. at 10 degrees C. per minute.

The cryovials are then stored in vapor phase of liquid nitrogen at −196°C.

Differentiating Culture Conditions

Three T75 flasks of 10-15 days old suspension cultures are spun down for5 min at 400 rpm. The cells are removed and placed into a 12 mlcentrifuge tube and spun down for 5 min at 600 rpm. The growth medium isremoved, and cells resuspended in fresh control media (no EGF or othermitogen) plus hormone mix. This step is repeated one more time to ensurethe complete removal of the mitogen from the media. Dissociated cellsare plated in media hormone mix at a density of 1×10⁶ cells/ml onpoly-L-ornithine-coated (15 μg/ml; Sigma) glass coverslips in 24-wellNunclon culture dishes with 0.5 ml/well. After 7 to 14 days in culture,cells will change morphology into a neuron-like or glial-like phenotype.Following staining with specific antibodies which recognize markers ofneural precursors, of neurons and of glia, the differentiation efficacyof the hormone mix is quantified. The density of positively stained cellbodies are determined in at least 20 randomly selected fields from eachculture dish or well using the 40× objective. For quantitation of totalNeuN-ir, GFAP and nestin-ir cells produced, a total of 3 experiments areperformed resulting in total of 6 culture dishes or wells analyzed foreach condition. Neural differentiation efficacy of a growth factor (orhormone mixture) is calculated as the percentage of NeuN-immunoreactivecells (relative to total number of cells in a dish identified with DAPInuclear stain).

Indirect Immunocytochemistry

Rabbit polyclonal antisera and mouse monoclonal antibodies directedagainst specific antigens are used as primary antibodies for indirectimmunofluorescence. Coverslips fixed with 4% paraformaldehyde for 20 minfollowed by three washes (10 min each) in phosphate buffer saline (PBS).After the PBS rinse, coverslips are processed for single or duallabeling and incubated with primary antibodies generated from differentspecies. The primary antibodies are made in PBS/10% normal goatserum+0.3% triton X-100. After 2 hours incubation at 37° C., thecoverslip is rinsed in PBS. Fluorescent conjugated secondary antibodies(1:100, 1:200, Jackson ImmunoResearch) are applied in PBS for 30 min atroom temperature. Coverslips are then washed three times (10 min each)in PBS, rinsed with water, placed on glass slides, and coverslippedusing Fluorsave (Calbiochem). Fluorescence is detected and photographedusing Zeiss Laser Scanning Confocal microscope (model LSM 510). Theprimary antibodies that are used are against: Nestin (1:1000;PharMingen), Vimentin (1:200, Boehringer), Glial Fibrillary AcidicProtein (1:500, Sigma), 04 (1:100, Chemicon), Myelin Basic Protein(1:200, Boehringer), and CNPase (1:500, Stemberger Monoclonals), NeuN(1:100, Chemicorp), β-tubulin class III (1:1000, Sigma), Neuron SpecificEnolase (1:100, Chemicon), human specific Neurofilament (1:150,Boehringer), MAP2 (1:200, Chemicon), GABA (1:5000, Sigma), CholineAcetyltransferase (1:200, Chemicon), Tyrosine Hydroxylase (1:4000,Incstar), Serotinin (1:200, Chemicon), type IV collagen (1:50, Dako),Laminin (1:300, Sigma), CD10 (1:100, PharMingen), muscle actin (1:1000,Sigma), HLA-DR (1:200, PharMingen), CD45 (1:100, PharMingen), Mac-1(1:100, Chemicon); alkaline phosphatase staining kit (Sigma #85L-2).

Flow Cytometric Analysis

To assess the actions of specific treatment on differentiation or toestablish a relative profile within a culture condition of differentcell populations labeled with the markers mentioned above, the harvestedcells are subject to Flow Cytometric Analysis. Cells are rinsed withfluorescence activated cell sorting (FACS) buffer (EBSS and 1% HIFBS)and 1×10⁶ cells are added to 100 μl of FACS buffer supplemented with theappropriate primary antibodies and incubated at 4° C. for 30 min. Afterwashing, secondary antibodies are added and incubated at 4° C. for 30min. For biotinylated antibodies, isotope controls are used to setgates; otherwise, gates are set with cells alone. Cell viability ismonitored using propidium iodide exclusion. Flow Cytometric Analysis isperformed with FACScan™ (Becton-Dickinson) with all events gated on theforward and side scatter.

Western Blotting

The culture is washed three times in cold phosphate buffered saline(PBS), scraped into ice-cold PBS, and lysed in ice-cold lysis buffercontaining 20 nM Tris/HCl (pH=8.0), 0.2 mM EDTA, 3% Nonidet P-40, 2 mMorthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mM NaCl, and10 μg each of aprotinin and leupeptin per ml. After incubation on icefor 10 min, the samples are centrifuged at 14,000×g for 15 min andsupernatants are collected. An aliquot is removed for total proteinestimation (bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sample is separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibody is blocked with 5% non-fat drymilk overnight at 4° C. Immunoblotting is carried out with theappropriate primary antibody followed by their corresponding peroxidaseconjugated secondary antibodies. The blots are developed by enhancedchemiluminescence method (ECL, Amersham).

Reverse transcription-polymerase chain reaction (RT-PCR) and NorthernAnalysis Total RNA is extracted using TRIzol (Life Technologies-BRL)according to the recommended protocol.

RT-PCR: aliquot of 1 μg of RNA is reverse-transcribed in the presence of50 mM Tris-HCL, pH 8.3, 75 mM KCL, 3 mM MgCl2, 10 mM DTT, 0.5 mM dNTPsand 0.5 μg Oligo-dT (12-18) (Pharmacia) with 200 U Superscript RnaseH-Reverse Transcriptase (Life Technologies-BRL). Aliquots of cDNAequivalent to 40 ng of total RNA are amplified in 25 μl reactionscontaining 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 50 pmol ofeach primer, 400 μM-dNTPs, and 0.5 U AmpliTaq DNA polymerase(Perkin-Elmer). The PCR thermal profile is determined for each pair ofprimer sequence used.

Southern blot. 15 μl aliquots of the amplified PCR products are run on a2% agarose Tris-acetate gel containing 0.5 μg/ml ethidium bromide. Thebands are transferred by capillary action to a Hybond-N+ membrane anddetected using the appropriate radiolabeled probes. The radioactivemembrane is exposed overnight to a BioMaxMR autoradiographic film(Kodak) at −80° C.

Northern hybridization. Aliquots (20 μg) of total RNA are fractioned onagarose formaldehyde gels. The RNA is transferred by capillary actionfrom the gel matrix to Hybond-N+ (Amersham) using 10×SSC, and fixed ontomembrane by baking. These membranes are hybridized with the adequateradio-labeled probes, washed with decreasing concentration of SSC, 0.1%SDS and exposed to a BioMaxMR autoradiographic film for 24 hours (Kodak)at −80° C.

Human Umbilical Cord Blood Contains Multi-Potent Progenitor Cells WhichGive Rise to Neural Lineage

General Methods Following the methods which are generally set forthabove, cord blood was shown to contain cells which can differentiate toneural cells.

Source of Cells

The mononuclear cells for this study were isolated from human umbilicalcord blood samples. The cord blood samples were obtained from the stumpof the umbilical cord on the placental side post-partum. Between 50 and100 ml of blood was obtained per procedure. Cells were spun down,resuspended in cryopreservative medium and frozen in liquid nitrogenuntil needed.

Handling of the Cells and Culture Media. The frozen cells were thawed,spun down, resuspended and plated in 75 mm culture flasks in minimalessential medium (DMEM) supplemented with 2 mM glutamine (100.times.stock from Gibco/BRL), 0.001% B-mercaptoethanol, 1× non-essential aminoacids (100× stock from Gibco/BRL) and 10% FBS (stem cell qualified,Gibco/BRL). After 24 to 72 hrs, the medium was replaced with serum free“Neural Proliferation Medium” which consisted of “N2” medium (DMEM/F121:1, Gibco/BRL) supplemented with 0.6% glucose, insulin 25 μg/ml,transferrin 100 μg/ml, progesterone 20 nM, putrescine 60 μM, seleniumchloride 30 nM, glutamine 2 mM, sodium bicarbonate 3 mM, HEPES 5 mM,heparin 2 μg/ml and EGF 20 ng/ml, bFGF 20 ng/ml. Differentiation mediumconsisted of the “Neural Proliferation Medium” without the EGF and bFGF,but instead containing retinoic acid (0.5 μM) plus 100 ng/ml nervegrowth factor (NGF). Detection of proliferating cells was accomplishedby incubating cultures with bromodeoxyuridine (BrdU) (5 μM) for 24-48hrs with subsequent visualization of BrdU immunoreactive cells.

Isolation of RNA. Total RNA was isolated from human cord blood cells (orfractions thereof) using the RNA STAT-60 kit using the protocolrecommended by the manufacturer (Tel-Test “B”, Inc. Friendswood, Tex.77546). Following RNA isolation, its OD density was measured at 260 nm,and stored at −80° C. Integrity was tested on 1% non-denaturing SeakemLE agarose gel (FMC Bioproducts, Rockland, Me.).

DNA Microarray

Total RNA was prepared as above. Total RNA obtained from human cordblood cells, with or without RA+NGF treatment from different batcheswere pooled together for this experiment (15 to 20 g total RNA wasneeded per chip). The human genome U95A array (HG-U95A) from AffymetrixInc. was used in this experiment. The single array represents about12,500 sequences. The experiment was done at the Functional GenomicsCore, Microarray Facility at the H. Lee Moffitt Cancer Center & ResearchInstitute using GeneChip Fluidics station 400, a GeneChip Hybridizationoven, and an HP GeneArray™ scanner. Analysis of the GeneChip microarrayhybridization pattern was performed using GeneChip Analysis Suite 4.0software.

Reverse Transcription (RT). RT was performed using random hexamers asprimers. Final volume was 20 μl with 1 μg of total RNA from eachfraction of cells. The reaction mixture contained 1 mM of eachdeoxynucleoside triphosphate (dNTP), 1 U/μl RNase inhibitor, 5 mM MgCl2,2.5 U/μl Murine leukemia virus (MuLV) reverse transcriptase, 2.5 μMrandom hexamers in 50 mM KCl and 10 mM Tris-HCl (pH 8.3). It was firstincubated at room temperature for 10 min, and then at 42° C. for 15minutes. The mixture was then heated at 99° C. for 5 minutes and cooledon ice for 5 min to inactivate the transcriptase.

Polymerase Chain Reaction (PCR). PCR was performed in the same tubes asRT, in 100 μl total volume. Final concentrations were 2 mM MgCl2, 0.2 mMof each dNTP, and 2.5 U/100 μl Ampli Taq DNA polymerase in the 50 mM KCland 10 mM Tris-HCl buffer (pH 8.3). For generation of various cDNAfragments, a PE 9700 thermocycler (Perkin Elmer, Foster City, Calif.)was programmed as follows: 1 cycle at 95° C. for 105 sec, 35 cycles at95° C. for 15 sec, followed by 60° C. for 30 sec, and finally 1 cycle at72° C. for 7 min. Both RT and PCR were done using Perkin Elmer's GeneAmpRNA PCR kit. To identify the presence of various neuronal markers,primers were constructed based on published human sequences. For Nestin(accession #X65964), forward primer: nt 2524-2542 and reverse primer: nt2921-2903. For Mushashi-1 (accession #AB012851), forward primer: nt319-339 and reverse primer: nt 618-598. For Necdin (accession#AB007828), forward primer: nt 2374-2393 and reverse primer: nt2767-2747. For Neurofilament subunit NF-L (accession #X05608), forwardprimer: nt 3155-3173 and reverse primer: nt 3521-3501. Primers wereselected using the SEQWEB (version 1.1) software available on the USFcomputer network.

Antibodies

The primary antibodies used included: Mushashi-1 (donated by Prof. H.Okano), Nestin (1:200, Chemicon); NeuN (1:100, Chemicon), class IIIβ-tubulin (1:200, Sigma); GFAP (1:500, Sigma), BrdU (1:400, Chemicon),MAP2 (1:200, Chemicon), pleiotrophin (1:400, R&D Systems).

Western Blot. Cultures were processed using standard methods forperformance of Western blot analysis using the following procedure.Cultures were washed three times in cold phosphate buffered saline(PBS), scraped into ice-cold PBS, and lysed in ice-cold lysis buffercontaining 20 nM Tris/HCl (pH 8.0), 0.2 mM EDTA, 3% Nonidet P-40, 2 mMorthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mM NaCl, and10 μg each of aprotinin and leupeptin per ml. After incubation on icefor 10 min, the samples were centrifuged at 14,000×g for 15 min andsupernatants were collected. An aliquot was removed for total proteinestimation (Bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sample was separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibody was blocked with 5% non-fat drymilk overnight at 4° C. The blots were analyzed using the Kodak DS 1DDigital Science Electrophoresis Documentation and Analysis System 120v.0.2.

Immunocytochemistry After 7-14 DIV, the cultures were fixed with 4%paraformaldehyde in 0.1 M phosphate buffer (PB) for 20 minutes. Thecultures were then washed 3 times with phosphate buffered saline priorto beginning immunocytochemistry.

Cell Counts For estimates of cell number in culture, 20 random visualfields (40× objective) in 4 culture dishes for each marker were viewed.The total number of cells visualized under phase contrast microscopy andthe number of positively labeled (immunoreactive) cells was counted ineach visual field. The mean number of labeled cells was then expressedas a percentage of the total number of cells per field.

Results

Cord blood cells, cultured in the presence and absence of retinoic acid(RA) and Nerve Growth Factor (NGF), gave rise to cells bearing neuralprogenitor markers as evidenced by profiles of gene and proteinexpression. A total of 322 genes were either up- or down-regulated by afactor of at least 2, evidenced by measurements using a human microarray“gene chip”. The greatest degree of up-regulation (44-fold increase) wasseen in the mRNA for neurite outgrowth extension protein orpleiotrophin. A significant degree of down regulation was seen in theexpression of tenascin (decreased 8.8-fold), an extracellular matrixprotein that inhibits neurite outgrowth in developing neuronal tissuesand in fibronectin (decreased 5.8-fold), an extracellular matrix proteinthat favors development of blood cell lineages. Other transcriptsassociated with neurogenesis that increased significantly (>2 fold)include glypican-4 (increased 4.9-fold), neuronal pentraxin II(increased 2.3-fold), neuronal growth associated protein 43 (increased2.7-fold); neuronal PAS1 (increased 2.3-fold). Mushashi-1 wasupregulated 1.5-fold. A selection of other genes associated withneurogenesis that were up- or down-regulated is listed in Table I.Concomitant with the increased expression of markers indicative ofneurogenesis, there was a decrease in expression of genes associatedwith hematopoiesis (Table II). The greatest changes occurred in theexpression of HLA class I locus C heavy chain, macrophage receptorMARCO, secreted T cell activation protein Attractin (attractin),leucocyte immunoglobulin-like receptor-8 (LIR-8), thymocyte antigenCD1c, erythropoietin receptor and erythropoietin.

In a parallel set of experiments, total RNA was extracted, and RT-PCRwas performed. The mRNA for nestin and necdin was identified in bothcontrol and RA+NGF treated cultures using primers based on publishedhuman sequences. In each case a product of appropriate length was seenon the gel (FIG. 1A for untreated cells and FIG. 1B for treated cells).Nestin is considered a marker of early neural development, but can alsobe seen in endothelial precursors. Necdin is the gene that codes forneuron specific nuclear protein. The m-RNA for Mushashi-1, the earliestmarker of neural precursors was detected in RA+NGF treated cultures andminimally detected in DMEM-treated controls. The mRNA for neuriteoutgrowth promoting protein (pleiotrophin) was detected in RA+NGFtreated cells, but the signal was much weaker in untreated cultures(FIG. 1C). The mRNA for glypican-4 was detected under both conditions.The mRNA for GFAP, a marker of astrological cells, was also detectedunder both conditions, though the signal was stronger in the RA+NGFtreated cells. No messenger RNA for neurofilament subunit NF-L wasdetected in either treated or untreated cells although it was seen to beup-regulated in the microarray. A negative RT control (without reversetranscriptase) was run with all the reactions to check for genomic DNAcontamination in the RNA preparation while human-actin primers(Clontech) were used as a positive control. The inventors also testedprimers for Mushashi-1 and Neurofilament subunit NF-L using human brainRNA (Clontech) by RT-PCR; these each generated a single band ofappropriate length (data not shown).

Microscopic examination of immunostained cultures treated with RA+NGFrevealed a heterogeneous mixture of cell types ranging from large flatepitheloid cells to small spindle-shaped cells with fine branchingneuritic processes. A significant proportion (5-10%) of the small cellsin the RA+NGF treated cultures, but not control cultures treated withDMEM, were Mushashi-1 immunoreactive (See FIG. 2A). A similar proportion(5%) of the small cells exhibited β-tubulin III immunoreactivity (FIG. 2B). Approximately 50% of the cells were immunoreactive for BrdU,indicating that the cells were continuing to proliferate (data notshown). Antibodies to nestin (purchased from Chemicon, raised againstrat nestin) failed to recognize the human form of nestin, though theyhad been shown to react with rat nestin in rat bone marrow-derivedneural progenitor cells (Sanchez-Ramos, et al., Neuroscience News 3,32-43, 2000). Approximately 50% of RA+NGF treated cells wereimmunoreactive for GFAP, a marker of astrocytes (FIG. 2E). Western blotsof the cultures confirmed the presence of Mushashi-1 protein,β-tubulin-III protein, pleiotrophin, GFAP and NeuN in both treated anduntreated cells. Densitometric analysis of the blots showed that NGF+RAtreatment increased protein expression (relative to β-actin) ofMushashi-1, β-tubulin-III, pleiotrophin and NeuN (Table III).

TABLE I Expression of Genes Associated with Neurogenesis Gene transcriptFold change following RA + NGF Neurite outgrowth-promoting protein +44extracellular matrix-associated protein that enhances axonal growth inperinatal cerebral neurons [Raulo, 1992 #384] Glypican-4 +4.9 glypican-4is expressed in cells immunoreactive for nestin and the D1.1 antigen,markers of neural precursor cells. Glypican-4 expression not detected inearly postmitotic or fully differentiated neurons [Hagihara, 2000 #375]β-tubulin folding cofactor D ~+4.6 Pro-galanin +3.9 Found in neurons ofarcuate nucleus of hypothalamus FE65 stat-like protein ~+3.8 the exon9-inclusive (E9) form is exclusively expressed in neurons[Hu, 1999 #369]Glial acidic fibrillary protein ~+3.2 Neuron derived orphan receptor~+2.2 Neuronal pentraxin II (NPTX2) ~+2.3 member of a new family ofproteins identified through interaction with a presynaptic snake venomtoxin taipoxin; may function during synapse formation and remodeling[Kirkpatrick, 2000 #303] Neuronal growth protein 43 (GAP-43) ~+2.7Identifies neurons, but also developing muscle cells [Moos, 1993 #395]Neuronal PAS1 (NPAS1) +2.3 transcription factors selectively expressedin the central nervous system [Zhou, 1997 #394] Neuronal DHP-sensitive,voltage-dependent, +2.1 calcium channel alpha-1D subunit Bonemorphogenetic protein 1 (BMP-1) +2 BMP-1/Tolloid is found at the neuralplate/ectodermal transition. Expression is maintained in thepremigratory neural crest, and transiently in the migrating cephalicneural crest cells. [Marti, 2000 #391] Retinal glutamate transporterEAAT5 ~+2 TrkC ~+2 Receptor for neurotrophin-3 (NT3) ENO2 gene forneuron specific (gamma) +1.9 enolase Human brain protein recognized bythe sera of ~+1.8 patients with paraneoplastic sensory neuronopathy Bonemorphogenetic protein 2A ~+1.8 Neuronal PAS2 (NPAS2) +1.7 transcriptionfactors selectively expressed in the central nervous system [Zhou, 1997#394] Survival motor neuron pseudogene +1.7 Glial Growth Factor 2 +1.6Neural cell adhesion molecule (N-CAM) Exon ~+1.6 SEC Follistatin-relatedprotein (FRP) +1.6 Microtubule-associated protein 2 (MAP2) ~1.6Vesicular acetylcholine transporter +1.6 Neurofilament subunit M (NF-M)+1.5 Neurofilament subunit NF-L +1.5 Musashil ~1.5 Bone morphogeneticprotein 11 (BMP11) +1.5 BMP-11 is expressed in the developing nervoussystem; at higher doses induces nervous tissue [Garner, 1999 #392]Tenascin-C ~8.8 Tenascin-C is an extracellular matrix protein thatinhibits neurite extension, and promotes cell proliferation andmigration [Thomas, 1996 #397; Anstrom, 1996 #398]

TABLE II Downregulation of Genes Associated with Development of BloodLines Fold decrease HLA class I locus C heavy chain −6.4 Macrophagereceptor MARCO −4.9 secreted T cell activation protein Attractin −3.6(attractin) alpha-1 collagen type II −3.0 Leucocyte immunoglobulin-likereceptor-8 −2.8 (LIR-8) Thymocyte antigen CD1c −2.5 Erythropoietinreceptor ~−2.6 Erythropoietin ~−2.4 Monocyte chemotactic protein-2 −2.1LAG-3 mRNA for CD4-related protein involved ~−2.3 in lymphocyteactivation Interleukin-7 receptor (IL-7) −2.2 Complement receptor type 1−2.1 T cell receptor −2 p50-NF-kappa B homolog −2 Lymphocyte-specificprotein tyrosine kinase ~−2 (LCK) LAG-3 mRNA for CD4-related proteininvolved ~−2.3 in lymphocyte activation Erythrocyte membrane proteinRh30A (Rhesus ~−2.1 antigen) Erythrocyte membrane protein band 4.2 ~−2.9(EPB42) Leukocyte IgG receptor (Fc-gamma-R) −1.8 Erythroblast macrophageprotein EMP −1.5

TABLE III Densitometric Measurement of Expressed Proteins Separated bWestern Blot Ratio to Ratio to MW β-Actin β-Actin Protein marker DMEMNGF % change Musashi-1 36 kD 0.805 0.93 +15.5% β-III tubulin 75-80 kD  0.328 0.50 +52.4% Pleiotrophin 18 kD 0.179 0.315 +75.9% GFAP 46 kD 0.5380.515 −4.3% NeuN 51 kD 0.6 0.715 +19.1% β-Actin 42 kD

Discussion of Results

These findings demonstrate that human umbilical cord blood containscells that can be induced to express markers of neural development,including Mushashi-1, glypican-4 and β-tubulin III. Recent work hasdemonstrated Mushashi-1 immunoreactivity in the developing and/or adultCNS tissues of frogs, birds, rodents, and humans (Kaneko, et al.,Developmental Neuroscience 22, 139-53 (2000). The anti-Mushashi-1monoclonal antibody has been shown to react with undifferentiated,proliferative cells of the sub-ventricular zone in the CNS of allvertebrates tested. Glypican-4, upregulated five-fold in the cord bloodcultures treated with RA+NGF, has been reported to be expressed in cellsimmunoreactive for nestin and the D1.1 antigen, other known markers ofneural precursor cells, but it has not been detected in earlypostmitotic or fully differentiated neurons (Hagihara, et al,Developmental Dynamics 219, 353-67 (2000). β-tubulin III is one of themost specialized tubulins specific for neurons (Fanarraga, et al.,European Journal of Neuroscience 11, 517-27 (1999). Both theupregulation and the post-translational processing of class-IIIβ-tubulin are believed to be essential throughout neuronaldifferentiation (Laferriere, et al., Cell Motility & the Cytoskeleton35, 188-99 (1996) and Laferriere, et al., Biochemistry & Cell Biology75, 103-17 (1997).

The cord blood cultures treated with RA+NGF also increased expression ofmany genes specific for neurons including pentraxin II, GAP43, FE65stat-like protein, neuronal PAS1 and PAS2. Neuronal pentraxin II is amember of a new family of proteins identified through interaction with apresynaptic snake venom toxin taipoxin. Neuronal-pentraxin-II mayfunction during synapse formation and remodeling (Kirkpatrick, et al,Journal of Biological Chemistry 275, 17786-92 (2000). Neuronal growthassociated protein 43 (GAP43) is considered a specific neuronal markerbut may also be expressed in developing myocytes (Moos, T. &Christensen, L. R. GAP43 identifies developing muscle cells in humanembryos. Neuroreport 4, 1299-302 (1993). FE65 stat-like protein (theexon 9-inclusive form) is specifically expressed in neurons (Hu, et al.,Journal of Neuroscience Research 58, 632-40 (1999). Neuronal PAS1 andPAS2 are transcription factors selectively expressed in the centralnervous system (Zhou, et al., Proceedings of the National Academy ofSciences of the United States of America 94, 713-8 (1997). Other genesindicative of neurogenesis that were expressed following treatmentincluded the neurofilament subunits-NF-L and NF-M, microtubuleassociated protein 2 (MAP2), the vesicular acetyl choline transporter,and neuronal DHP-sensitive, voltage-dependent, calcium channel alpha-1Dsubunit. Cord blood cells expressed mRNA for neuronal specific enolase,but this protein is also expressed by many cells in bone marrow,especially megakaryocytes. The greatest change observed in cord bloodcultures treated with RA+NGF was a 44-fold increase in expression ofmRNA for an extracellular matrix-associated protein that enhances axonalgrowth in perinatal cerebral neurons (Raulo, et al., Journal ofBiological Chemistry 267, 11408-16 (1992). At the same time, there was asignificant decrease in expression of mRNA for tenascin, anextracellular matrix protein which inhibits neurite outgrowth (Kukekov,et al., Experimental Neurology 156, 333-44 (1999). There was alsoevidence for glial cell development. Increased expression of the glialcell marker GFAP was measured in the microchip data, and confirmed byimmunocytochemistry. Concomitant with the increased expression ofmarkers indicative of neurogenesis, there was a decrease in expressionof genes associated with development of blood cell lines.

The present findings provide evidence that cord blood contains amulti-potent cell capable of differentiating into a neural lineage. Theease with which the umbilical cord blood can be obtained, stored, andexpanded in culture could make this a preferable source of cells fortransplantation for neurodegenerative diseases, gene delivery to thecentral nervous system, and repair of brain and spinal cord injuries.

Example

Identification and Isolation of Mononuclear Cells Expressing Neuronal,Astrocytic or Oligodendrocytic Markers and Use of Mononuclear Cells toEffect Transplantation in Stroke

This series of experiments is directed to using both cell culturetechniques and an animal model of cerebral ischemia to establish humancord blood as a viable source of NSCs for the treatment of CNS diseaseor injury. These studies determine the existence of mononuclear cells incord blood that express neuronal, astrocytic or oligodendrocytic markersand identify those mononuclear cells that give rise to neural celllineages. The cord blood stem cells are shown to provide a stable,readily available source of NSCs which become functional neurons and arecapable of producing behavioral recovery at a comparable level to thatobserved with transplantation of fetal neurons.

General Methods.

Culture Media.

The frozen cells are thawed, spun down and resuspended and plated in 75mm culture flasks in minimal essential medium (DMEM) supplemented with 2mM glutamine (100× stock from Gibco/BRL), 0.001% B-mercaptoethanol, 1×non-essential amino acids (100.times. stock from Gibco/BRL) and 10% FBS(stem cell qualified, Gibco/BRL). After 24 to 72 hrs, the medium isreplaced with serum free “Neural Proliferation Medium” which consists ofN2 medium (DMEM/F12 1:1, Gibco/BRL) supplemented with 0.6% glucose,insulin 25 μg/ml, transferrin 100 μg/ml, progesterone 20 nM, putrescine60 μM, selenium chloride 30 nM, glutamine 2 mM, sodium bicarbonate 3 mM,HEPES 5 mM, heparin 2 μg/ml and EGF 20 ng/ml, bFGF 20 ng/ml.Differentiation medium consists of the “Neural Proliferation Medium”without the EGF and bFGF, but instead containing retinoic acid (0.5 μM)plus a specific growth factor (NGF, BDNF, or GDNF)

Transfection of Cord Blood Cells with Fluorescent Green Protein Drivenby the Mushashi-1 Promoter.

An ΔE1 adenovirus bearing hGFP under the control of the Mushashi-1promoter (AdP/Mushashi) (generously donated by H. Okano of Japan) areused to transfect umbilical cord blood cells. This adenoviral DNA vectoris a plasmid DNA that contains a portion of the viral genome in whichthe E1 A region is deleted and the hGFP under control of the Mushashi-1promoter has been inserted in the place of the E1A region of the genome.Cells to be transfected are plated in 0.5 ml of serum-free “NeuralProliferation Medium”. To each culture dish of cells to be transfected0.8 μg of the DNA is diluted and mixed into 50 μl of Opti-Mem® I ReducedSerum Medium Without Serum (Life Technologies, Inc). Eight μl of PlusReagent Mix is added and incubated at room temperature for 15 min.Lipofectin Reagent (Life Tech, Inc) is diluted and mixed in a secondtube (0.5 μl into 50 μl of Opti-Mem I Reduced Serum Medium WithoutSerum). After 30 min incubation at room temperature, the pre-complexedDNA is mixed with diluted Lipofectin Reagent and incubated for 15 min atroom temperature. Then the DNAPlus-Lipofectin Reagent complexes (100 μl)are added to each well and mixed gently by rocking the plate back andforth. The cultures are incubated at 37° C. in 5% CO2 for 4-5 h. After24 to 48 hrs, selected cultures are harvested to assess efficiency oftransfection.

Isolation of RNA.

Total RNA is isolated from human cord blood cells (or fractions thereof)using the RNA STAT-60 kit using the protocol recommended by themanufacturer (Tel-Test “B”, Inc. Friendswood, Tex. 77546). Following RNAisolation, its OD density is measured at 260 nm, and stored at −80° C.Integrity is tested on 1% non-denaturing Seakem LE agarose gel (FMCBioproducts, Rockland, Me.).

Reverse Transcription (RT).

RT is performed using random hexamers as primers. Final volume is 20 μlwith 1 μg of total RNA from each fraction of cells. The reaction mixturecontains 1 mM of each deoxynucleoside triphosphate (dNTP), 1 U/μl RNaseinhibitor, 5 mM MgCl2, 2.5 U/μl Murine leukemia virus (MuLV) reversetranscriptase, 2.5 μM random hexamers in 50 mM KCl and 10 mM Tris-HCl(pH 8.3). It will first be incubated at room temperature for 10 min, andthen at 42° C. for 15 minutes. The mixture will then be heated at 99° C.for 5 minutes and cooled on ice for 5 min to inactivate thetranscriptase.

Polymerase Chain Reaction (PCR).

PCR is performed in the same tubes as RT, in 100 μl total volume. Finalconcentrations are 2 mM MgCl2, 0.2 mM of each dNTP, and 2.5 U/100 μlAmpli Taq DNA polymerase in the 50 mM KCl and 10 mM Tris-HCl buffer (pH8.3). For generation of various cDNA fragments, a PE 9700 thermocycler(Perkin Elmer, Foster City, Calif.) is programmed as follows: 1 cycle at95° C. for 105 sec. 35 cycles at 95° C. for 15 sec, followed by 60° C.for 30 sec, and finally 1 cycle at 72° C. for 7 min. Both RT and PCR aredone using Perkin Elmer's GeneAmp RNA PCR kit. To identify the presenceof various neuronal markers, primers are constructed based on publishedhuman sequences. For Nestin (accession #X65964), forward primer: nt2524-2542 and reverse primer: nt 2921-2903. For Mushashi-1 (accession#AB012851), forward primer: nt 319-339 and reverse primer: nt 618-598.For Necdin (accession #AB007828), forward primer: nt 2374-2393 andreverse primer: nt 2767-2747. For Neurofilament subunit NF-L (accession#X05608), forward primer: nt 3155-3173 and reverse primer: nt 3521-3501.Primers were selected using the SEQWEB (version 1.1) software availableon the USF computer network.

Western Blot.

Cultures are washed three times in cold phosphate buffered saline (PBS),scraped into ice-cold PBS, and lysed in ice-cold lysis buffer containing20 nM Tris/HCl (pH=8.0), 0.2 mM EDTA, 3% Nonidet P-40, 2 mMorthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mM NaCl, and10 μg each of aprotinin and leupeptin per ml. After incubation on icefor 10 min, the samples are centrifuged at 14,000×g for 15 min andsupernatants are collected. An aliquot is removed for total proteinestimation (Bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sample is separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibody is blocked with 5% non-fat drymilk overnight at 4° C.

MCAO Induction.

Sprague Dawley rats are anesthetized with isofluorane and an incisionmade from the caudal end of the sternomastoid and sternothyroid musclesextending toward the ears. Using blunt dissection techniques, the rightcommon carotid artery is exposed and carefully dissected free of thevagus nerve. The external carotid will then be tied off and an embolus(a 40 cm length of 4.0 monofilament) is inserted through the externalcarotid approximately 25 mm into the internal carotid. At this point,the embolus is blocking the origin of the right middle cerebral artery.The embolus is left in place for 1 hr. After removal, the externalcarotid is cauterized and the incision closed. The animals are allowedto recover for 24 hr prior to transplantation.

Transplantation.

The freshly isolated MNCs are resuspended in HBSS+15 mM HEPES at a cellconcentration of 100,000 cells/4 The coordinates for the injection siteare 1.2 mm anterior and +2.7 mm lateral to the bregma and −5.2 and −4.7mm ventral to the dura with the toothbar set at zero. Five microlitersof the cell suspension are deposited at 2 sites in the striatum adjacentto the infarct site along a single needle tract. Each injection of 2.5μl is delivered at the rate of 1 μl/min. The needle is left in place foran additional 5 min after the injection and then withdrawn slowly. Theincision is closed with wound clips. For the transplantation of theexpanded and/or differentiated MNCs, the cells are lifted from theculture flasks with gentle mechanical trituration or lifted with trypsin(0.25%) and 1 mM EDTA at 37 C for 3-4 min and washed three times withHBSS+15 mM HEPES. Cell concentration is adjusted to 100,000 cells/μl.

Behavioral Testing Methods.

Twenty-four hours after stroke, the animals undergo a standardizedneurological screening exam measuring 5 motor and postural activities toverify the extent of the MCAO damage. This battery is repeated at onemonth post stroke. In addition, the animals are tested at both timepoints in the Passive Avoidance test of learning and memory. In theacquisition phase of the test, the animal is placed on a platform in thecorner of a Plexiglas cage. When it steps off the platform, the rat willreceive a scrambled foot shock (approximately 2 mA) for as long as itremains off the platform. Learning is measured by the amount of timerequired for the rat to remain on the platform continuously for 3minutes, and the number of times it leaves the platform. Twenty-fourhours later, retention is measured by placing the rat on the platform,and recording the latency to step-down measured to a maximum of 3 minand the number of step-downs. Animals are also tested in the RotorodTest of motor coordination. The animal is placed on a revolving rod (16rpm) and the latency to the first fall as well as the number of falls ina 3 minute test is recorded. The test is repeated twice for a total of 3tests per testing session with a minimum 30 min. rest between tests. Thethird behavioral observation includes Spontaneous Activity Monitoring.The animals are placed in a square acrylic box overnight with aninfrared grid to measure movement and direction. The Elevated Body SwingTest, a measure of motor asymmetry is also performed. The animal is heldby the base of the tail and lifted 2″ above the base of the cage. Thedirection that the head and body is lifted is recorded. The test isrepeated 20 times. The final test is Skilled Forepaw Use. This is also ameasure of motor asymmetry. The animal is placed in an acrylic chamberwith two descending staircases. Each step is baited with 5 food pellets.The chamber is designed such that each staircase can only be reached byone paw. The number of pellets retrieved measures the function of eachlimb. There is a 5 day training period, during which the animals arepartially food deprived. All behavioral data are reported asmean+/−.sem.

Tissue Preparation in Culture Preparations.

After 7-14 DIV, the cultures are fixed with 4% paraformaldehyde in 0.1 Mphosphate buffer (PB) for 20 minutes. The cultures are then washed 3times with phosphate buffered saline prior to beginningimmunocytochemistry.

Tissue Preparation of Brain Sections.

The rats are sacrificed under deep chloral hydrate (10%) anesthesia andtranscardial perfusion of the brain with 50 ml of 0.1 M phosphate buffer(PB) and then 250 ml 4% paraformaldehyde in 0.1 M PB performed. Thebrain is removed, post-fixed for 24 hr and cryopreserved in 20% sucroseprior to cutting 30 μm thick frozen sections through the forebrain.

Immunohistochemistry.

Single and double immunofluorescence histochemistry are performed.Briefly, the floating sections will first be quenched by incubation in a10% methanol, 3% hydrogen peroxide solution in phosphate buffered saline(PBS) followed by pre-incubation in 10% normal serum (horse or goat;Vector) in 0.3% Triton-X100 (Sigma) in PBS. The sections are transferredto primary antibody in 2% normal serum, 0.3% Triton X-100/PBS andincubated overnight at 4° C. The primary antibodies that are usedinclude: Mushashi-1 (donated by Prof. H. Okano), Nestin (1:200,Chemi-Con); vimentin (1:500 Chemi-Con) as markers of early neuralprecursors; NeuN (1:100, Chemi-Con) and Hu (1:20 Molecular Probes) classIII β-tubulin (1:200, Sigma) to identify human neurons at specificstages of development; human specific GFAP (1:200, StembergerMonoclonals) to identify astrocytes; and 04 or 2′3′ cyclic nucleotide 3′phosphodiesterase (CNPase, 1:200, Sigma) to identify oligodendrocytesderived from the transplanted human MNCs. The sections are then washedin PBS before being placed in secondary antibody conjugated to eitherfluorescein or rhodamine for 2 hours. The sections are rinsed in PBS,mounted and coverslipped with Vectashield. Confirmation that a cell isdoubly-stained with be obtained by z-stacking analysis of imagescollected with a Zeiss Confocal Microscopes (LSM 510).

Cell Counts.

For assessment of cell number in culture, 20 random visual fields (40×objective) in 4 culture dishes for each marker in 3 replicates areviewed. The total number of cells and the number of positively labeledcells are counted. For each experimental condition, the number ofpositive cells and the total number of cell nuclei stained with4′,6-dimidinee-2′-phenylindole dihydrochloride (DAPI) are determined.The total counts are then expressed as a percentage of the totalDAPI-stained nuclei. For quantification of immunofluorescence in brainsections, an unbiased counting methodology are used. Neurons aredirectly counted in a small number of sections at predetermined uniformintervals for the entire set of sections containing specific CNS nuclei.Within each section to be counted the field of view is focused at thetop of the section using a 40× objective. The focus is then shiftedthrough the section and the number of positive profiles not present atthe top of the section is counted.

Analysis.

The number of animals to be used in these studies was based on a poweranalysis of data obtained in previous experiments in this laboratory.The analysis showed that a minimum of 10 animals per group is needed tofind a difference in the variables of interest at a significance levelof p<0.05. All quantifiable results are expressed as mean+/−sem and areanalyzed using Analysis of Variance (ANOVA). All post-hoc tests areconducted using a Scheffe test.

Identification and Isolation of Stem/Progenitor Cells Present inUmbilical Cord Blood

The ideal way to identify and isolate neural progenitor cells among theheterogeneous population of mononuclear cord blood cells is to utilize acell surface marker to which a fluorescent or magnetic bead antibody tagis attached to facilitate sorting and separation. Unfortunately, bothNestin and Mushashi-1 are located in the cytoplasm. Surface-basedimmunoselection strategies do not yet permit the prospectiveidentification or specific extraction of neural stem/progenitor cells. Anovel strategy has been used to identify and monitor internal molecularmarkers of neural progenitor cells and to separate the neuralprogenitors from other cells using fluorescence activated cell sorting(FACS) (N. S. Roy et al., Journal of Neuroscience Research 59, 321-31(2000)). This method relies on coupling the promoters required forneuroepithelial-specific gene expression with a reporter gene (eitherlacZ or Green Fluorescent Protein-GFP). More specifically,cis-regulatory elements (the “promoter”) required for the expression ofMushashi-1 or .alpha.-tubulin-1 were placed upstream to the reportergene GFP (Wang et al., Nature Biotechnology 16, 196-201 (1998) and N. S.Roy et al., Journal of Neuroscience Research 59, 321-31 (2000)) Usingthis approach, neural progenitors and young neurons have been identifiedand selectively harvested from a variety of heterogeneous samples,including both adult and fetal mammalian forebrains at differentdevelopmental stages (Wang, et al., supra, and Keyoung et al., Societyfor Neuroscience Abstract, 159 (2000)).

Experimental Design.

The inventors identify and separate neural progenitor cells by FACS ofcord blood cells transfected with the gene for GFP, driven by theneuronal promoter α-tubulin-1 (Tα1) or by the Mushashi-1 promoter.Mononuclear cells are obtained from the placental stump of the umbilicalcord after delivery and processed by Ficoll centrifugation (See GeneralMethods). This results in nearly 100% recovery of mononuclear cells.These cells are cryopreserved in aliquots of 2 million cells until theyare to be used. After thawing, and plating in culture flasks insupplemented minimal essential medium (DMEM) plus FBS 10% for 48 hrs,the medium is changed to “Neural Progenitor Proliferation Medium” for 2days (See Methods for definition of the media). Then, the mononuclearcells are transfected in a suspension culture with a plasmid or viralvector containing the gene for GFP under the control of P/Tα1 orMushashi-1 (See General Methods for details on Transfection techniqueand description of the vectors). After a 6-hour transfection, the cellsare spun down, resuspended in “Neural Progenitor Proliferation Medium”and plated in small culture flasks (See General Methods). GFP shouldtypically be expressed by appropriate target cells within 2 days oftransfection. Flow cytometry and sorting of GFP+ cells are performedafter 2-7 days in culture. Cells are washed, dissociated and analyzed bylight forward and right-angle (side) scatter, and for GFP fluorescencethrough a 510.+−.20 nm band pass filter as they traverse the beam of theLaser (488 nm, 100 mW). Sorting is done using a purification-modealgorithm. Cells detected as being more fluorescent than background aresorted at 1,000-3,000 cells/s. Sorted GFP+ cells are plated in 24 wellculture plates in “Neural Proliferation Medium” (See General Methods fordetails) and BrdU. At 2 and 7 days post-FACS, the sorted cultures arefixed and immunostained for BrdU together with either Mushashi-1,β-tubulin-III, Nestin, NeuN, MAP2, glial fibrillary acidic protein(GFAP) or O4 (to detect oligodendrocytes).

Results and Alternative Method for Enriching Stem-Like Cells.

Cells transfected with the plasmid DNA encoding P/Tα1:GFP or the viralvector encoding P/Mushashi:GFP identify neural progenitors and immatureneurons as evidenced by immunoreactivity for Mushashi-1, β-tubulin-III,and Nestin. In most instances, at least 50% of the Mushashi-1(+) cellsare co-labeled with BrdU antibody indicating that the cells areproliferating. Based on the work of Wang et al., supra, manyMushashi1-driven GFP+ cells should be labeled for up to 7 to 10 days.These neural progenitors and their daughters should be selected andsubstantially enriched by FACS. Further, they also showed that 0.36% ofadult ventricular zone dissociates expressed Tα1-driven GFP. Assumingthat only 0.01% of cord blood cells express Mushashi1-driven GFP, andthe transfection efficiency using the plasmid DNA is 12.5%, theinventors estimate that for every 5×10⁶ cord blood cells processed, theinventors shall obtain 625-1000 neural precursor cells. However, usingan adenoviral vector results in a much greater transfection efficiency.For that reason, the inventors also use a.DELTA.E1 adenovirus bearinghGFP under the control of the Mushashi promoter (AdP/Mushashi:hGFP)provided generously by H. Okano of Japan.

In an alternative method, the inventors first identify the leastcommitted stem-like cells from the mononuclear cells in the cord bloodand isolate this population from both CD34+ and CD34− cells prior toinducing proliferation and promoter-based isolation of neuralprogenitors. The basic premise of this strategy is that stem cells arequiescent and express very few cell surface markers, except during aproliferation phase. Primitive stem cells fail to stain with Hoescht33342 and Pyronin Y and can be separated on this basis using FACS.Further, separation of cells that express P-gp, the transmembraneprotein product of the multiple drug resistance gene (MDR), which islikely to be expressed in cells that exhibit characteristics of stemcells, could also be performed. These staining characteristics could beused to separate stem cells from cord blood mononuclear cells usingFACS. If FACS demonstrates that P-gp+ cells are also cells that fail tostain with the fluorescent dyes (Hoescht 33342 and Pyronin Y), thenmagnetic bead cell sorting is used to physically separate the P-gpimmunoreactive cells from cord blood. In order to increase the yield ofneural progenitor cells, it is preferable to start with the smallerpopulation of the least committed cells found in cord blood.

Assessing the Self-Renewal Capacity of the Neural Progenitor Population

In the example above, the inventors identify a subpopulation of cellsenriched with neural progenitor cells or uncommitted stem cells. Itwill, therefore, be critical to expand the cell populations in order toobtain sufficient cells for study and eventually for transplantation. Inthis study, the inventors will determine whether there are differencesin the ability of the isolated populations of cells to proliferate andthe best agents for inducing proliferation in the neural progenitors.

Experimental Design.

Mononuclear cells are obtained and the subpopulations isolated asdescribed above. The inventors will focus primarily on the GFP+ cellscontaining neural progenitors. These cells are plated in Corning T75flasks with “Neural Progenitor Proliferation Medium”. This serum-free,defined medium contains epidermal growth factor (EGF) and basicfibroblast growth factor (bFGF) and is used to induce proliferation ofneural stem cells derived from fetal or adult brains. Once the culturesreach confluence (about 1 week), the cells are lifted by incubation with0.25% trypsin, and 1 mM EDTA for 3-4 minutes. An aliquot of cells isreplated with BrdU to assess the proportion of cells that are activelyproliferating. The cells are replated after 1:3 dilution with NeuralProgenitor Proliferation medium. Cell yield and viability is alsodetermined with the trypan blue dye exclusion assay after each passage,for at least five passages.

Results.

Neural stem cells proliferate with exposure to EGF or bFGF and thecombination of these growth factors optimally allow for the continuous,rapid expansion and passaging of human neural stem cells. Alternatively,there is an extensive list of trophic factors and cytokines that may bemore or less effective in inducing proliferation. These include othermembers of the EGF family such as transforming growth factor (TGF)−,amphiregulin, betacellulin and heregulin; FGF2 and the related FGF1 andFGF4, platelet-derived growth factor family (PDGF), interleukins, andmembers of the TGF β superfamily. There may be some degree ofdifferentiation that occurs despite culturing in presence of knownmitogens. The proportion of cells that continue to proliferate(determined by the BrdU assay before each passage) will guide us in theselection of the optimal mitogens for the neural progenitors.

Assessing the Capacity of Cord Blood Derived Neural Progenitors toDifferentiate into Neurons, Astrocytes or Oligodendrocytes In Vitro.

The mononuclear cells which proliferate subpopulations of mononuclearcells as established above can differentiate into neurons, astrocytesand oligodendrocytes. The inventors have shown that a small populationof non-hematopoietic stem cells in the bone marrow stromal cell fractionwill differentiate into neurons and astrocytes. Moreover, preliminaryresults show that cord blood treated with RA+NGF for less than one weekexpress a marker seen in early neuronal development, β-tubulin-III. Inthis study, the inventors demonstrate that treatment with“Differentiation Media” will drive the neural progenitors into neuronaland glial phenotypes.

Experimental Design.

Mononuclear cells from the first or second passage of the subpopulationswith the greatest proliferative capacity as determined above (GFP+cells) are replated in 35 mm culture dishes in the presence of a“neuronal differentiation medium” (See Methods for definition) and aseries of specific neurotrophic factors. The first to be tried are brainderived neurotrophic factor (BDNF, 10 ng/ml) since this media has beenused previously with bone marrow stromal cells to differentiate thecells along neural lineages. After 7-14 days in vitro (DIV), culturesare processed for Western blotting, RT-PCR and immunocytochemistry toidentify cells that express neural markers. The markers to be examinedinclude nestin, vimentin, glial fibrillary acidic protein (GFAP) tolabel astrocytes, O4, myelin basic protein and CNPase to identifyoligodendrocytes and NeuN, β-tubulin class III, Hu, Neuron-SpecificNuclear antigen (NeuN), human specific neurofilament and microtubuleassociated protein (MAP-2) to identify neurons. Quantification isdescribed in the General Methods.

Results.

Neural markers are observed in the subpopulations chosen for assay basedon preliminary results with cord blood and results obtained withdifferentiation of bone marrow stromal cells. Further, the populationmost likely to give rise to these neural cells is the GFP-expressingcells (driven by the Mushashi-1 promoter). Alternatively, it may benecessary to use the stem-like cells obtained by selecting the leastcommitted of cells from the cord blood, nonetheless the inventors arestill able to obtain significant numbers of differentiated neurons andglia following treatment with differentiation media.

Example

Expanded Population of Mononuclear Cells and Expression of NeuralMarkers after Transplantation into Middle Cerebral Artery Occlusion(MCAO) Model of Stroke

In addition to demonstrating the existence of the neural phenotype invitro, it is important to show that the isolated and differentiatedcells could express or maintain their neural phenotype aftertransplantation. The culture environment is easily controlled andmanipulated. The environment into which the cells are transplanted invivo is much less predictable; and there are many influences on thecells once they are placed in situ, some of which may alter theproliferative capacity or phenotypic lineage of the cell. Therefore, theinventors demonstrate that these stem-like cells maintain the ability tobecome neurons, astrocytes or oligodendrocytes in the brain.

Experimental Design.

Sprague Dawley rats (n=10/group) are assigned to one of the followinggroups: 1) Middle cerebral artery occlusion (MCAO); 2) MCAO with astriatal transplant of freshly isolated mononuclear cells; 3) and 4)MCAO+expanded GFP+ cells from the two subpopulations with the highestproliferative capacity as determined above; 5) and 6)MCAO+expanded/minimally differentiated GFP+ cells as determined above.The untreated cord blood cells in group 2 are labeled with thefluorescent dye PKH26 for later identification in the brain and thentransplanted into the striatum in the penumbral region of the infarct.(The isolated neural progenitors will already be labeled by the GFP).The animals are evaluated on a series of behavioral measures and aneurological exam at 24 hr and one month. This includes two paradigmsthe inventors have used to demonstrate behavioral deficits after strokeand recovery following transplantation, the passive avoidance test ofcognitive function and the rotorod test of motor coordination. Theanimals will then be perfused with 4% paraformaldehyde and the brainsharvested for histological and immunohistochemical analysis of graftsurvival and neural differentiation. Sections are examined for thepresence of PKH26-positive (or GFP+) cells, and cells that express humanNuclear Matrix Antigen (NuMA), allowing a second method of identifyinghuman cord blood-derived cells in the rat brain). Other sections aredouble-labeled for PKH26 or NuMA and Hu, class III β-tubulin, NeuN,GFAP, O4. The proportion (%) of cord blood derived cells (NuMA-ir andPKH26+) that express neuronal markers (NeuN, Hu, or class IIIβ-tubulin), glial markers (GFAP) and oligodendrocyte markers (O4) aredetermined.

Results.

Based on the preliminary results the inventors see both behavioralimprovements and surviving stem-cell progeny in the MCAO-injured brain.Behavioral improvements are observed in all transplant groups, althoughit is better in the expanded/minimally differentiated cells where theexpanded cell population have already been committed to a neural lineageand therefore may be expected to express more neurons. With the wholeMNC cell fraction, there are fewer of the subpopulation cells that giverise to neural cells than in the expanded subpopulations.

Example

Effect of Transplantation of Human Umbilical Cord Mononuclear Cells inStroke

In a small series of pilot studies, the cord blood mononuclear cellswere transplanted into the striatum of animals that had either undergonea permanent or temporary (1 hr) middle cerebral artery occlusion (MCAO).The cells (500,000 cells/implant) were transplanted immediately uponthawing or were treated in culture for a week with various trophicfactors (BDNF, NGF, EGF+bFGF) prior to transplantation. Preliminaryresults obtained from the temporary stroke model revealed differencesbetween the groups on the rotorod test of motor coordination. Animalswhich received the retinoic acid+NGF treated mononuclear cells were ableto stay on a rotating axle longer and fell off fewer times in the 3minute test period than did all other animals in the study. This studyevidenced that the umbilical blood cells provide a novel cell source fortransplantation in stroke which can improve function.

Example

Parenteral administration of Cord Blood Fractions in the Treatment ofNeurological Damage from Ischemia (Stroke)

Methods and Materials

1. HUCB Sources and Preparation:

HUCB was provided and analyzed by Cryocell international, INC. The cellscontain 77.2% 95% CD34+ cells, respectively. The specimen was stored inliquid nitrogen and the cells were restored at 37° C. Aftercentrifugation at 1000 rpm/min for 5 min at 4° C., the cells were washedwith 0.1 M PBS. Nucleated HUCB were counted using a cytometer to ensureadequate cell number for transplantation. The final dilution isapproximately 3×10⁶ HUCB in 500 μl saline for injection in each rat.

2. Animal MCAo Model:

Adult male Wistar rats (n=38) weighing 270-300 g were employed in allexperiments. Briefly, rats were initially anesthetized with 3.5%halothane and maintained with 1.0-2.0% halothane in 70% N20 and 30% 02using a face mask. Rectal temperature was maintained at 37° C.throughout the surgical procedure using a feedback-regulated waterheating system. Transient MCAo was induced using a method ofintraluminal vascular occlusion modified in the laboratory [Chen, etal., J Cereb Blood Flow Metab 1992; 12(4): 621-628]. The right commoncarotid artery, external carotid artery (ECA) and internal carotidartery (ICA) were exposed. A length of 4-0 monofilament nylon suture(18.5-19.5 mm), determined by the animal weight, with its tip rounded byheating near a flame, was advanced from the ECA into the lumen of theICA until it blocked the origin of the MCA. Two hours after MCAo,animals were anaesthetized with halothane and reperfusion was performedby withdrawal of the suture until the tip cleared the lumen of the ECA.

3. In Vitro-Chemotaxis Assay

1) Ischemia Brain Tissue Extracts:

Animals were sacrificed at 6 h, 24 h and 1 w (n=3 per time point) afterthe onset of MCAo; a normal control group (n=3) was employed in whichthe animals were not subjected to surgical procedures. Tissue extractswere obtained from the experimental rats and control rats. Forebraintissues were immediately obtained from interaural 12 mm to interaural 2mm [Paxinos et al, The Rat Brain in Stereotaxic Coordinates. AcademicPress, San Diego. 1986]. Each specimen was dissected on a bed of iceinto hemispheres ipsilateral right side and contralateral to the MCAo.The tissue sections were homogenized by adding IMDM (150 mg tissue/mlIMDM) and incubated on ice 10 min. The homogenate was centrifuged at100,000 g for 20 min at 4° C. and the supernatant extracted.

2) Ischemia Brain Tissue Extract Assay on HUCB Migration

Chemotactic activity of ischemia brain tissue extracts toward HUCB atdifferent time points was evaluated by using 48-well micro chemotaxischamber technique, as described [Xu et al, Hematology, 4:345-356, 1999]with some modification. HUCBs were resuspended in IMDM (serum free) at10⁶ cells/ml. Twenty-five microliters of tissue extracts prepared fromnormal and ischemic brain at 6 h, 24 h and 1 w after MCAo (150 mgtissue/ml IMDM) were placed in the lower chamber of the 48-well microchemotaxis chamber. A polycarbonate membrane (8 μm pore size) strip wasplace over the lower wells and 50 μl of HUCB suspension (1×10⁶ cells/ml)was place in each of the upper wells. Migration of HUCBs was allowed for5 h at 37° C. incubation and the number of migrated cells into the lowerwells was then measured.

4. In Vivo-Treatment with HUCB:

Experimental Groups:

Group 1 (Control): MCAo alone without donor cell administration (n=10);Group 2: 3×10⁶ human UCB cells injected intravenously at 24 h after MCAo(n=6); The animals of group 1, 2 were sacrificed at 14 days after MCAo.In order to test the effects of delayed (7 day) treatment, the inventorsincluded two additional groups. Group 3 (Control): MCAo alone withoutdonor cell administration (n=5) and rats were sacrificed at 35 daysafter MCAo; Group 4: 3.times.10.sup.6 HUCB cells were injectedintravenously at 7 days after MCAo and rats were sacrificed at 35 daysafter MCAo (n=5).

Implantation Procedures:

At 1 or 7 days post-ischemia, randomly selected animals received HUCB.Animals were anesthetized with 3.5% halothane and then maintained with1.0-2.0% halothane in 70% N20 and 30% 02 using a face mask mounted in aKopf stereotaxic frame. Approximately, 3×10⁶ HUCB cells in 0.5 ml totalfluid volume were injected into a tail vein.

Functional Tests:

In all animals, a battery of behavioral tests were performed beforeMCAo, and at 1, 7, 14, 21, 28, 35 days after MCAo by an investigator whowas blinded to the experimental groups. The battery of tests consistedof:

1) Rotarod test: An accelerating rotarod was used to measure rat motorfunction [Hamm R J, J Neurotrauma 11(2): 187-196 1994; and Chen, J Med;31(1-2):21-30, 2000]. The rats were placed on the rotarod cylinder andthe time the animals remained on the rotarod was measured. The speed wasslowly increased from 4 rpm to 40 rpm within 5 min. A trial ended if theanimal fell off the rungs or gripped the device and spun around for twoconsecutive revolutions without attempting to walk on the rungs. Theanimals were trained 3 days before MCAo. The mean duration (in seconds)on the device was recorded with 3 rotarod measurements one day beforesurgery. Motor test data are presented as percentage of mean duration(three trials) on the rotarod compared with the internal baselinecontrol (before surgery).

2) Adhesive-removal somatosensory test [Schallert, Brain Res 379(1):104-111 1986; Hernandez, Exp Neurol, 102(3): 318-324 1988; Zhang, NeurolSci, 174(2): 141-146, 2000; and Chen, Neuropharmacology, 39(5): 711-7162000]. Somatosensory deficit was measured both pre- and postoperatively.All rats were familiarized with the testing environment. In the initialtest, two small pieces of adhesive-backed paper dots (of equal size,113.1 mm²) were used as bilateral tactile stimuli occupying thedistal-radial region on the wrist of each forelimb. The rat was thenreturned to its cage. The time to remove each stimulus from forelimbswas recorded on 5 trials per day. Individual trials were separated by atleast 5 min. Before surgery, the animals were trained for 3 days. Oncethe rats were able to remove the dots within 10 seconds, they weresubjected to MCAo.

3) Modified Neurological severity score (mNSS): [Borlongan, Brain Res;676(1): 231-234 1995; Shohami, Brain Res, 674(1): 55-62, 1995; Chen,Neurotrauma. 1996; 13(10):557-568 1996; Shaller Adv Neurol, 73:229-238,1997]. Neurological function was graded on a scale of 0 to 18 (normalscore 0; maximal deficit score 18). mNSS is a composite of motor,sensory, reflex and balance tests [Germano, J Neurotrauma; 11(3):345-3531994]. In the severity scores of injury, one score point is awarded forthe inability to perform the test or for the lack of a tested reflex;thus, the higher score, the more severe is the injury.

5. Histological and Immunohistochemical Assessment:

Animals were allowed to survive for 14 or 35 days after MCAo, and atthat time animals were reanaesthetized with ketamine (44 mg/kg) andxylazine (13 mg/kg). Rat brains were fixed by transcardial perfusionwith saline, followed by perfusion and immersion in 4% paraformaldehyde,and the brain, heart, liver, spleen, lung, kidney and muscle wereembedded in paraffin. The cerebral tissues were cut into seven equallyspaced (2 mm) coronal blocks. A series of adjacent 6 μm-thick sectionswere cut from each block in the coronal plane and were stained withhematoxylin and eosin (H&E). The seven brain sections were traced usingthe Global Lab Image analysis system (Data Translation, Malboro, Mass.).The indirect lesion area, in which the intact area of the ipsilateralhemisphere was subtracted from the area of the contralateral hemisphere,was calculated [Swanson, J. Cereb Blood Flow Metab, 10(2): 290-293,1990]. Lesion volume is presented as a volume percentage of the lesioncompared to the contralateral hemisphere.

Single and double immunohistochemical staining [Li, Brain Res, 838(1-2):1-10, 1999] was used to identify cells derived from HUCB. Briefly, astandard paraffin block was obtained from the center of the lesion,corresponding to coronal coordinates for bregma −1˜1 mm. A series of 6μm thick sections at various levels (100 μm interval) were cut from thisblock and were analyzed using light and fluorescent microscopy (Olympus,BH-2). To detect the distribution of transplanted HUCB cells in otherorgans (i.e. heart, liver, lung, spleen, kidney and muscle, bonemarrow), 3 sections (6 μm thick, 100 μm interval) from each organ wereobtained and numbers MAB1281 reactive cells measured. MAB1281 (MouseAnti-human nuclei monoclonal antibody, Chemicon International, Inc) ismarkers for human [Vescovi, et al., Exp Neurol; 156(1):71-83 1999].After deparaffinization, sections were placed in boiled citrate buffer(pH 6.0) within a microwave oven (650-720 W). After blocking in normalserum, sections were treated with the monoclonal antibody (mAb) againstMAB 1281 diluted at 1:300 in PBS with FITC staining for identificationHUCB. Analysis of MAB1281 positive cells is based on the evaluation ofan average of 10 histology slides of brain, 3 slides from each organ perexperimental animal.

To visualize the cellular co-localization of MAB1281 andcell-type-specific markers in the same cells, fluorescein isothiocyanateconjugated antibody (FITC, Calbiochem, Calif and red cyanine-5.18) wasemployed for double-label immunoreactivity. Each coronal section wasfirst treated with the primary MAb1281 mAb with FITC staining foridentification HUCB. As described above, and were followed withcell-type-specific antibodies, a neuronal nuclear antigen (NeuN forneurons, dilution 1:200; Chemicon, Calif), microtubule associatedprotein 2 (MAP-2 for neurons, dilution 1:200; Boehringer Mannheim) andglial fibrillary acidic protein (GFAP for astrocytes, dilution 1:1000;Dako, Calif.) and FVIII (Von Willebrand Factor, dilution: 1:400; Dako)with CY5 staining. Negative control sections from each animal receivedidentical preparations for immunohistochemical staining, except thatprimary antibodies were omitted. A total of 500 MAb1281 positive cellsper animal were counted to obtain the percentage of MAb1281 cellscolocalized with cell type specific markers (MAP-2, NeuN, GFAP, FVIII)by double staining.

Laser Scanning Confocal Microscopy (LSCM):

Colocalization of MAB1281 with neuronal (NeuN, MAP-2, GFAP) andendothelial cell (FVIII) markers were conducted by LSCM using a Bio-RadMRC 1024 (argon and krypton) laser-scanning confocal imaging systemmounted onto a Zeiss microscope (Bio-Rad, Cambridge, Mass.) [Zhang Z G,1999] For immunofluorescence double-labeled coronal sections, green(FITC for HUCB) and Red cyanine-5.18 (Cy5 for MAP-2, NeuN or GFAP)fluorochromes on the sections were excited by a laser beam at 488 nm and647 nm; emissions were sequentially acquired with two separatephotomultiplier tubes through 522 nm and 680 nm emission filters,respectively. Areas of interest were scanned with a 40× oil immersionobjective lens in 260.6×260.6 m format in the x-y direction and 0.5 m inz direction.

6. Statistical Analysis:

The behavior scores (rotarod test, adhesive-removal test and NSS) wereevaluated for normality. Repeated measures analysis of variance wasconducted to test the treatment by time interactions, and the effect oftreatment over time on the behavior score. If an interaction oftreatment by time or overall treatment effect were significant at the0.05 level, the subgroup analysis would be conducted for the effect oftreatment at each time point at level 0.05. Otherwise, the subgroupanalysis would be considered as exploratory. The means (STD) and p-valuefor testing the difference between treated and control groups arepresented.

To evaluate the chemotactic activity of HUCB migration, counts of intactcells were performed on the normal brain tissue extracts, and ischemicbrain tissue extracts at 6 h, 24 h and 1 week of ischemic onset. Theinventors tested the normality and equal variances of each outcomemeasure. Data transformation or permutation tests would be considered,if data were ill behaved. The HUCB migration active were evaluatedbetween normal tissue and ischemic tissues, respectively. The maineffect was significant at level 0.05, then subgroup analysis would beconsidered with a significant effect at level of 0.05. The means (std)are reported.

Results

Functional Tests:

Rats treated with HUCB cells at 24 h after stroke showed no treatment bytime interaction for each treatment group on each neurobehavioral score(p-value for interactions >0.13). The overall treatment effect wassignificant on NSS with p<0.01, adhesive-removal test p=0.04 and rotarodtest p=0.01. FIGS. 3A, 3B and 3C shows that treatment at one day afterMCAo with HUCB significantly improved functional recovery at 14 days asevidenced by rotarod, adhesive-removal test and NSS scores (p<0.05).Rats treated with HUCB at 7 days after stroke showed no treatment bytime interaction on neurobehavioral scores with p-value for interactionat 0.88 for NSS, 0.41 for the adhesive-removal test and 0.09 for therotarod test scores. The overall treatment effect was significant onlyon NSS with p<0.05, and no treatment effect on the other tests (p=0.15for adhesive removal test and 0.55 for rotarod test score) was detected.FIG. 4A, 4B, 4C show treatment at 7 days after MCAo with HUCBsignificantly improved functional on NSS test (p<0.05) at 28 day and 35day after MCAo compared to control group. However, rotarod andadhesive-removal tests failed to show a significant difference comparedto control animals.

Histology:

Within the 6 μm thick coronal sections stained with H&E, dark and redneurons were observed in the ischemic core of all rats subjected to MCAowith and without donor transplantation at 14 and 35 days after MCAo. Nosignificant reduction of volume of ischemic damage was detected in ratswith donor treatment at 24 h and 7 days after ischemia, compared withcontrol rats subjected to MCAo alone. Within the brain tissue,identification of HUCB was characterized by MAB1281 staining. HUCBsurvived and were distributed throughout the damaged brain of recipientrats [FIG. 5]. MAB1281 reactive cells were observed in multiple areas ofthe ipsilateral hemisphere, including cortices and striatum of theipsilateral hemisphere. The vast majority of MAB1281 reactive cells werelocated in the ischemic boundary zone [FIG. 5]. Few cells were observedin the contralateral hemisphere. The data indicate that HUCB cellsdelivered to brain via an intravenous route preferably migrate into theinjured tissue. Some MAB 1281 positive cells encircle vessels, and somecells were detected in the nuclei of the capillary endothelial cellssurrounding the injury area [FIG. 5].

Double staining immunohistochemistry of brain sections revealed thatsome MAB1281-positive cells were reactive for the astrocyte marker GFAP,neuronal markers NeuN and MAP-2, for endothelial cell marker FVIII. Thepercentage of MAB1281 labeled expressed GFAP, NeuN, MAP-2 and FVIIIproteins was (about.6) %, (about.3) %, (about.2%) and (about.8%),respectively.

Ischemia Brain Tissue Extract Assay on HUCB Migration:

HUCB cells migrate in the presence of normal brain tissue and ischemictissue obtained at 6 h, 24 h and 1 w after MCAo. A significant increasein HUCB migration activity was detected in the presence of ischemiccerebral tissue harvested at 24 h after the onset of stroke (p<0.01). Atrend of increase in HUCB migration activity was apparent on tissueharvested at 6 hours and 1 week after MCAo (p>0.09) compared to HUCBmigration activity measured in the presence of on normal non-ischemicbrain tissue.

Results/Conclusions

The above-described experiments reveal that at 14 days and 35 days aftertransplantation, intravenously injected HUCB were found in the brain,and significantly more MAB 1281 positive cells were found in theipsilateral hemisphere than in the contralateral hemisphere. Many cellsmigrated into the boundary zone of ischemic brain and some cellssurrounded vessels. HUCB survive, and some express of cell-type-specificmarker GFAP, NeuN and MAP-2. Most important, a significant improvementin functional outcome on motor, sensory and modified NSS tests was foundin animals given HUCB intravenously at 1 day after stroke. In vitro, thedata showed there was significant HUCB migration activity in thepresence of ischemic cerebral tissue harvested at 24 h after MCAo(p<0.01) compared to normal non-ischemic brain tissue. The HUCBtreatment at ischemia 24 h promoted more HUCB migration into ischemicbrain that may facilitate to functional recovery after MCAo.

In this study, it is shown that intravenous infusion of HUCB enterbrain, survive, differentiate and reduce neurological deficits afterstroke. In the study, a small percentage of HUCB cells expressedproteins phenotypic of neuronal-like cells. Functional recovery wasfound within days after administration HUCB.

It was also shown that more HUCB were found in the lesioned hemispherethan in the intact Hemisphere as well as that ischemic brain tissueextracts induced migration of HUCB, suggesting that ischemia inducedchemotactic factors facilitate UCB migration.

The results described herein show that HUCB treatment at 24 h after MCAoin the present studies produced significant improved functional recovery(motor rotarod, somatosensory adhesive-removal test and NSS scores)after stroke. Treatment with HUCB at 7 days after MCAo showed functionalrecovery only on NSS test after MCAo. However, rotarod and somatosensoryadhesive-removal test did not show significant recovery. The treatmentbenefit of HUCB, thus, may depend on the time of treatment. Thetreatment benefit may be interrelated to the migration activity of HUCB.A significant increase in HUCB migration activity was detected in thepresence of ischemic cerebral tissue harvested at 24 h after MCAo.Treatment with HUCB at ischemic early may promote HUCB migration intoischemic brain and facilitate functional recovery after MCAo.

Almost 25% of cord blood harvests rapidly give rise to awell-established layer of fibroblastoid (MIC) cells. The rapid growth ofthese cells seems to be sustained by a population of (self-renewing)quiescent (G)) cells. MPC have large ex vivo expansion capacity as wellas on their differentiation potential cord blood-derived MPCs can bevisualized as attractive targets for cellular or gene transfertherapeutic options.

In conclusion, the experiments presented have shown that intravenouslyadministrated HUCB survive, migrate and improve functional recoveryafter stroke. Although the mechanism is unclear, the describedexperiments support the use of umbilical cord blood derived neural cellsfor the treatment of stroke.

Example

Parenteral Administration of Human Umbilical Cord Blood in ReducingNeurological Deficits After Traumatic Brain Injury

Materials and Methods

Preparation of Human Umbilical Cord Blood for Injection. The humanumbilical cord blood used was a gift from Cryocell International, INC.(Clearwater, Fla.). The specimen was stored in liquid nitrogen and thecells were restored at 37° C. After centrifugation at 1000 rpm/min for10 min at 4° C., the supernatant was removed and the cells were washedwith 0.1 M PBS two times. 30 μl of the cell suspension was mixed with 30μl of 0.4% trypan blue stain and the number of the viable cells wascounted with a hemacytometer and a counter under a phase contrastmicroscope. The total number of the harvested cells was calculated andthe final dilution was 2×10⁶ cells in 300 μl saline.

Controlled Cortical Injury Animal Model and the Injection of HUCB.Wistar rats were anesthetized with 350 mg/kg body weight chloralhydrate, intraperitoneally. Rectal temperature was controlled at 37° C.with a feedback regulated water-heating pad. A controlled corticalimpact device was used to induce the injury. Rats were placed in astereotactic frame. Two 10 μm diameter craniotomies were performedadjacent to the central suture, midway between lamda and bregma. Thesecond craniotomy allowed for movement of cortical tissue laterally. Thedura was kept intact over the cortex. Injury was induced by impactingthe left cortex (ipsilateral cortex) with a pneumatic piston containinga 6 mm diameter tip at a rate of 4 m/s and 2.5 mm of compression.Velocity was measured with a linear velocity displacement transducer.

Twenty-four rats subjected to TBI were divided into three groups.Experimental group (n=8): 24 hours after TBI, rats were slowly injectedover a 10 minute duration with 2×10⁶ cells in 300 ml saline via a tailvein. Placebo control group (n=8): 24 hours after TBI, rats were slowlyinjected over a 10 minute duration with 300 ml saline via a tail vein.TBI only group (n−8): the rats only were subjected to TBI and notreatment. All rats were killed 28 days after the treatment.

Tissue Preparation.

(1) Paraffin sections: Four animals from each group were euthanized withan overdose of ketamine and xylazine administered intraperitoneally andperfused with intra-cardiac heparinized saline followed by 10% bufferedformalin. The brains, hearts, lungs, livers, kidneys, spleens, muscleand bone marrow were removed and stored in 10% buffered formalin for 24hours. Seven standard 2 mm thick blocks were cut on a rodent brainmatrix and then embedded with paraffin. Two millimeter thick blocks ofthe other organs were also cut and embedded with paraffin. A series ofadjacent 6 mm thick sections were cut and a section of each block of thebrain and other organs was stained with H&E. Standard H&E staining wasemployed for morphological analysis under light microscopy. (2)Vibratome sections: An additional four rats from each group received theintravenous administration of 1 ml of saline containing fluoresceinisothiocyanate (FITC)-dextran (50 mg/ml, 2×10⁶ molecular weight; Sigma,St. Louis, Mo.). This dye circulated for 1 min, after which theanesthetized rats were killed by decapitation. The brains were rapidlyremoved from severed heads and placed in 4% paraformaldehyde at 4° C.for 48 hr. Coronal sections (100 μm) were cut on a vibratome.

Immunohistochemistry.

Single staining was performed for identification of HUCB cells using aprimary mouse anti-human nuclei monoclonal antibody (MAB1281) andsecondary Cy5-conjugated F (ab′)2 Fragment rabbit anti-mouse IgG in thecoronal sections of all organs. Double staining was also performed oncoronal cerebral sections. Brains sections were initially stained forneuronal markers, NeuN and MAP-2, or an astrocytic marker, glialfibrillary acidic protein (GFAP), with the correspondence primaryantibodies and the secondary FITC-conjugated F (ab′)2 fragment, andsubsequently double stained with primary MAB1281 antibody and secondantibodies of Cy5-conjugated-F(ab′)2 fragment for identification ofhuman umbilical cord blood cells. Briefly, 6 m thick sections from TBI,TBI+saline and TBI+HUCB groups were deparaffinized and the sections wereput in boiling citrate buffer (pH=6) in a microwave oven for 10 min foridentification of neurons. After cooling at room temperature, thesections were incubated in 0.1% saponin-PBS at 4° C. overnight for mAbNeuN (dilution 1:400, Chemicon) and MAP-2 (dilution 1:400, Chemicon).Antimouse FITC-conjugated F (ab′)2 fragment (dilution 1:20, Calbiochem,Calif.) was then added and incubated for one week. To identifyastrocytes, the sections were treated with 0.1% pepsin 37° C. for 15 minand then pAb GFAP (dilution 1:400, Dakopatts) was added. The sectionswere incubated with antirabbit FITC-conjugated F (ab′)2 fragment(dilution 1:20, Calbiochem, Calif) for one week. The above sectionsstained with FITC-conjugated F (ab′) fragment were subsequentlyprocessed for identification of a human cellular nuclei antigen with aprimary mouse anti-human nuclei monoclonal antibody, MAB1281 (dilution,1:200) and a Cy5-conjugated F (ab′)2 fragment rabbit anti-mouse IgG(dilution, 1:20). The slides were analyzed using a fluorescentmicroscope (Olympus, BH-2). Negative control sections from each animalreceived identical staining preparation, except that the primaryantibodies or the secondary antibodies were omitted.

Three-dimensional image acquisition. In order to observe the relation ofthe donor's cells with the cerebral vessels, the vibratome sections wereanalyzed with a Bio-Rad (Cambridge, Mass.) MRC 1024 (argon and krypton)laser-scanning confocal imaging system mounted onto a Zeiss microscope(Bio-Rad). With the FITC-perfused tissue samples from each group, 10vibratome sections from interaural 6.38 mm to interaural 1.0 nun(Paxions and Watson, 1986) at 2 mm interval were screened at 488 nmunder a 10× objective lens. Sections stained with the MAB antibody (Cy5)were excited by a laser beam at 647 nm.

Estimates of Cell Number.

For measurement of MAB 1281 reactive cells, an average number of fiveequally spaced slides (approximately 100 p. m interval) were obtainedfrom each brain block and MAB 1281 reactive cells were counted withinthe seven 2 mm thick blocks encompassing the forebrain. Nine slides fromeach of these blocks were first stained with FITC staining foridentification NeuN (3 slides), MAP-2 (3 slides) and GFAP (3 slides),and were followed by Cy5 staining for identification of HUCB cells. Thenumber of the MAB 1281 reactive cells expressing NeuN, MAP-2 and GFAPwere counted, respectively, using fluorescent microscopy within allseven blocks. In order to reduce biases introduced by samplingparameters, all sections for MAB 1281 identification from rats werestained simultaneously. The criteria for MAB 1281 positive cells weredefined before the cells were counted by observers blinded to theindividual treatment. All MAB 1281 reactive cells were countedthroughout the coronal sections.

Neurological Functional Evaluation.

Neurological motor measurement was performed using an acceleratingRotarod-motor test. The rats were placed on the accelerating Rotarodtreadmill (Lab-line instruments, INC) and the rat's task was to walk andmaintain its equilibrium on the rotating rod that rotates at a graduallyincreasing speed. When the rat falls off the rod, a plate trips and aliquid crystal records the endurance time in seconds. All rats werepre-trained with five trials (warm up trials) performed daily for 3 daysprior to TBI to ensure stable baselines. After TBI and TBI followingadministration of HUCB or saline, the rats were tested on days 1, 4, 7,14 and 28 until sacrifice. The motor test data are shown as a percentageof an average of five trials on the rotarod test compared with theinternal baseline values.

Twenty-four hours after TBI or administration of HUCB or saline, allrats were evaluated using the neurological severity scores (NSS). NSS isa composite of the motor (muscle status, abnormal movement), sensory(visual, tactile and proprioceptive) and reflex tests. One point wasgiven for failure to perform a task. Thus, the higher score, the moresevere is injury, with a maximum of 14 points. Rats were reevaluated ondays 1, 4, 7, 14 and 28 after the treatment. All measurements wereperformed by observers blinded to individual treatment.

Statistical Analysis.

NSS and Rotarod tested scores were measured before injury and at 1, 4,7, 14 and 28 days after TBI. The numbers of MAB 1281 reactive cells werecounted at 28 days after treatment. The inventors were primarilyinterested in the effect of HUCB on the recovery of NSS. The analysisbegan by testing the difference in means of NSS between the two controlgroups. If there was no difference between the two controls at 0.05level, the two control groups were combined to increase the power. Theanalysis of covariance for ANOVA (repeated measures) was conducted totest the treatment by time interactions, and the effect of treatmentover time. If an interaction of time by time was detected at 0.10 level,then the subgroup analysis was conducted for the effect of treatment ateach time point at level 0.05. Otherwise, the subgroup analysis wasconsidered as exploratory. The same analysis approach was used toanalyze the outcome of Rotarod test score. Paired-t test was used totest the difference in means of cell counts between the injuredhemisphere and the control hemisphere.

Results

Histological analysis of organs. Sections from the blocks of brain andorgans were stained with H&E staining for the general histopathologicalevaluation. The architectural integrity of all organs analyzed underlight microscopy was not disrupted except for the initial mechanicalinjury of the brain. Bleeding, invasion of white cells, inflammatoryresponse and neoplasm were not observed on any slides aside from brain.

Distribution of MAB 1281 positive cells. No MAB 1281 positive cells wereobserved in the slides from only TBI and TBI+saline groups which did notreceive the injection of HUCB. Large numbers of MAB 1281 positive cellswere found in the vessels of the brain, heart, lung, liver, kidney,spleen, muscle and even bone marrow of the rats receiving the injectionof HUCB. A few scattered MAB 1281 positive cells were found in theparenchyma of these organs. In brain, MAB 1281 labeled cells wereobserved in the boundary zone of the injured area, cortex, striamm andcorpus callosum of the ipsilateral hemisphere. The MAB 1281 positivesignals were detected in the nuclei of the capillary endothelial cellssurrounding the injured area. Using laser confocal microscopy, theimplanted cells were confirmed to be integrated into sprouting vesselsin the boundary zone of the injured area. The total number of MAB 1281positive cells migrating into the parenchyma of both the ipsilateral andcontralateral hemispheres of the brain was counted and analyzed in theTBI+HUCB group. The numbers of MAB 1281 positive cells in theipsilateral hemisphere (43,597.+−.4265) were significantly greater thanthose in the contralateral hemisphere (13,742.+−.6471, p<0.05). The dataindicate that HUCB cells delivered to brain via an intravenous routepreferably migrate into the injured tissue.

Phenotypical Identification of MAB 1281 reactive cells. Doublefluorescent staining showed that some MAB 1281 positive cells expressedneuronal markers, NeuN and MAP-2, and an astrocytic marker, GFAP. Thesedouble-labeled cells were observed only in the ipsilateral hemispheresof the rats in the HUCB treated group. Most of these positive cells werelocated in the boundary zone of the injured area. 6.9.+−.1.3% of MAB1281 labeled cells in the ipsilateral hemispheres in the HUCB treatedgroup expressed NeuN. 5.8.+−.2.4% expressed MAP-2 and 9.7+−2.8%expressed GFAP. These data demonstrate that some implanted cells expressneuronal and astrocytic phenotypes.

Neurological and Motor Function Evaluation.

Two days after TBI, significantly lower scores of Rotarod test andsignificantly higher scores of NSS in three groups compared topre-injury were found. Rotarod Test scores were significantly improvedin TBI+HUCB group (138.0−+11.3% and 155.2−+16.2%) when compared with TBI(118.5.+−.17.0% and 129.2.+−.12.2%) and TBI+saline group (117.2+−13.6%and 133.2.+−.10.7%. p<0.05) at days 14 and 28 aRer administration ofHUCB. The neurological severity scores were also significantly improvedin TBI+HUCB group (4.2+−1.3 and 3.+−.0.8) when compared with TBI group(7.5.+−0.1.73 and 6.3.+−.1.3) and TBI+saline group (7.3.+−.0.9 and5.75.+−.0.9), p<0.05) at days 14 and 28 after the injection. The resultsindicate that intravenous administration of HUCB 24 hours after TBIreduce the motor neurological functional deficits caused by TBI.

CONCLUSIONS

The major findings of the above-described experiments were: (1) HUCBcells injected intravenously enter brain by day 28 after HUCB celladministration; (2) intravenous injection of HUCB reduces motor andneurological deficits by days 14 and 28 after administration; (3) thecells migrating into the parenchyma of the brain express the neuronalmarkers, NeuN and MAP-2, and the astrocytic marker, GFAP; (4) HUCB cellsintegrated into the vascular wall within target organ; (5) these cellsare also present in other organs and primarily localize to the vessels,without any obvious adverse effects. The data suggest that intravenousadministration of HUCB may be useful in the treatment of TBI.

These data demonstrate that a few injected cells migrate into theparenchyma of the brain, heart, lung, kidney, liver, spleen, muscle andbone marrow. Because the study was designed to measure the effect ofHUCB administered intravenously on traumatic brain injury, the numbersof HUCB cells (MAB 1281 positive cells) present in the cerebralparenchyma were counted and analyzed in the TBI+HUCB group.Significantly more MAB 1281 positive cells were found in the ipsilateralhemisphere than in the contralateral hemisphere. This indicates that theinjected cells preferably migrate into the injured hemisphere,especially to the boundary zone of the injured area after TBI and thatnearly all of the MAB 1281 positive cells expressing NeuN, MAP-2 andGFAP were located in the ipsilateral hemisphere, demonstrating that themicro-environment of the brain after injury may benefit the induction ofthe differentiation of HUCB stem cells into the neural cell phenotype.

Two tests (Rotarod test and NSS) were used to measure the neurologicalbehavioral responses to experimental injury in rats. The Rotarod Test isa well-established procedure for testing limb motor coordination andbalance aspects of motor performance in rats. The NSS is similar to theRotarod Test and is an economical, simple and rapid test for assessingmild motor, sensory and reflex deficits after TBI. These two tests aregenerally used for the evaluation of the effects of the drugs on thebehavioral responses after TBI and stroke in animals. Fourteen andtwenty-eight days after intravenous administration of HUCB, theneurological behavioral deficits were significantly reduced in the ratssubjected to TBI in the above-described experiments. These data indicatethat intravenous administration of HUCB can effectively improve theneurological outcome in rats after TBI and that intravenousadministration of HUCB to patients suffering damage to the brain and/orspinal cord represents a viable therapeutic approach for treating suchinjuries, including traumatic brain injury.

While the invention has been described hereinabove, care should be takennot to limit the invention in a manner which is unintended and isinconsistent with the invention as set forth in the following claims.

What is claimed is:
 1. Neural cells obtained by exposing pluripotentstem or progenitor cells obtained from umbilical cord blood to an amountof a differentiation agent effective for changing a phenotype of thestem or progenitor cells to a neural phenotype.
 2. The neural cells ofclaim 1, wherein the differentiation agent is selected from the groupconsisting of retinoic acid, fetal or mature neuronal cells, BDNF, GDNF,NGF, FGF, TGF, CNTF, BMP, LIF, GGF, THF, IGF, CSF, KIT-SCF, interferon,triiodothyronine, thyroxine, erythropoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signaling molecules and mixtures thereof.
 3. The neuralcells of claim 1, wherein the differentiation agent is a mixture ofretinoic acid and NGF.
 4. A method of producing neural cells fromumbilical cord blood comprising: (a) obtaining a sample of mononuclearcells from the umbilical cord blood; and (b) growing the mononuclearcells from step (a) in a culture medium containing an effective amountof a differentiation agent for a period sufficient to change a phenotypeof the stem or progenitor cells to a neural phenotype.
 5. The method ofclaim 4, wherein the differentiation agent is selected from the groupconsisting of retinoic acid, fetal or mature neuronal cells, BDNF, GDNF,NGF, FGF, TGF, CNTF, BMP, LIF, GGF, THF, IGF, CSF, KIT-SCF, interferon,triiodothyronine, thyroxine, erythropoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signaling molecules and mixtures thereof.
 6. The methodof claim 4, wherein the differentiation agent is a mixture of retinoicacid and NGF.
 7. The method of claim 5, wherein the neuronal cells areselected from the group consisting of mesencephalic cells and striatalcells.
 8. A method of producing neural cells from umbilical cord bloodcomprising: (a) obtaining a sample of mononuclear cells from theumbilical cord blood; (b) selecting for and isolating a sample ofpluripotent stem or progenitor cells within the sample; and (c) growingthe stem or progenitor cells from step (b) in a culture mediumcontaining an effective amount of a differentiation agent for a periodsufficient to change a phenotype of the stem or progenitor cells to aneural phenotype.
 9. The method of claim 8, wherein the selecting andisolating step (b) is carried out using a magnetic cell separator toseparate out cells containing a CD marker.
 10. The method of claim 8,wherein the differentiation agent is selected from the group consistingof retinoic acid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF,TGF, CNTF, BMP, LIF, GGF, THF, IGF, CSF, KIT-SCF, interferon,triiodothyronine, thyroxine, erythropoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signaling molecules and mixtures thereof.
 11. The methodof claim 10, wherein the retinoic acid is selected from 9-cis retinoicacid, all trans retinoic acid and mixtures thereof.
 12. The method ofclaim 8, further comprising: (a) subjecting the mononuclear cells to anamount of an anti-proliferating cell agent effective to eliminateessentially all proliferating cells from the mononuclear cell sample;(b) exposing the remaining non-proliferating cells from step (a) to amitogen to provide a population of differentiated cells and quiescentcells comprising a population of pluripotent stem or progenitor cells;(c) growing the population of the differentiated cells and quiescentcells from step (b) to selectively grow the quiescent cells to anessential exclusion of differentiated cells.
 13. The method of claim 12,wherein the anti-proliferative cell agent is Ara-C.
 14. The methodaccording to claim 12, wherein the mitogen is selected from the groupconsisting of epidermal growth factor and pokeweed mitogen.